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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

The available two-generation reproductive toxicity studies with the read across substance in rats, indicate no concern for reproductive toxicity for the test substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Exposure period: about 18 wk.
P0 and P1:
Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
- Duration of test: P0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Details on study schedule:
Groups of 25 male and 25 female Sprague-Dawley rats were administered the test substance (purity 49.9%) at levels of 0, 500, 2000 or 4000 ppm in their diet over a period of 10 wk before mating, 2 wk during mating, and until after weaning of the pups (total period 18 wk). From every litter one or two pups were selected to again obtain 25 animals per sex and dose group for the F1 generation. These animals were similarly treated as the parent (P1) animals throughout premating, mating, pregnancy until sacrifice, after weaning of F2 progeny.
Remarks:
Doses / Concentrations: 0, 500, 2000 or 4000 ppm (equivalent to 250, 1000 and 2000 a.i. ppm) of test substance
Remarks:
F0 generation - 16-25, 61-101, 123-208 mg/kg bw/day (equivalent to 8-13, 31-51, 62-104 mg a.i./kg bw/day); F1 generation - 24-34, 96-123, 202-252 mg/kg bw/day (equivalent to 12-17, 48-62, 101-126 mg a.i./kg bw/day)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
For details, kindly refer to the attached background material section of the IUCLID.
Parental animals: Observations and examinations:
- Examination of P0 and P1 generation:
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. Males and females were paired for a 2-wk period, until mating was obtained. The P1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
- During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
Litter observations:
Examination of F1/P1 generation:
- On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1/P1 generation, which comprised 25 males and 25 females per group. The F1/P1 animals were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Sexual development of both males and females was assessed.
- Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous locomotor activity was also evaluated when the animals were between 7 and 8 wk old.
- After sexual maturity, F1 male and F1 female animals were paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
- Reflex development was assessed at designated time-points.
Postmortem examinations (parental animals):
Terminal examination of P0 and P1 animals:
- After weaning of their respective progeny, P0 and P1 parent males and females were sacrificed. Designated organs were weighed for P0 and P1 parents, as well as brain, spleen and thymus of one pup per sex per litter of each generation.
- Epididymal and testicular sperm parameters were evaluated for both P0 and P1 males.
- A macroscopic post-mortem examination was performed on all P0 and P1 parent males and females. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all P0 and P1 parents of the control and high dose groups.
- Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Postmortem examinations (offspring):
- A macroscopic post-mortem examination was performed on three pups per sex and per litter of each P0 and P1 (F1 and F2 generation) females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a macroscopic post-mortem examination. In all pups, the macroscopic lesions were preserved.
Statistics:
For details, kindly refer to the attached background material section of the IUCLID.
Reproductive indices:
For details, kindly refer to the attached background material section of the IUCLID.
Offspring viability indices:
For details, kindly refer to the attached background material section of the IUCLID.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 and 250 ppm, a marginally to slightly lower body weight gain was noted over all the dosing period for the males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period for the males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.

Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed (with lower spleen weights).
- At 1000 ppm, no effects were observed on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.
As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation respectively.
For further details, see under Any other information on results incl. tables.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 16-25 mg/kg bw/day (8-12.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: equivalent to 61-101 mg/kg bw/day (30.5-50.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 1000 ppm, a marginally to slightly lower mean body weight gain was noted.
- At 250 ppm, a marginally to slightly lower mean body weight gain was observed in both sexes. This was associated with lower liver weights of parental males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 1000 and 250 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period in both sexes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, reduced liver weights were observed.
- At 1000 and 250 ppm, lower liver weights in parental animals were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, necropsy of these animals revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents).
- At 1000 ppm, necropsy of parents revealed dilatation of the cecum in a single animal. 
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted at histopathological examination of sexual organs.
Histopathological findings: neoplastic:
not examined
Details on results:
See under Any other information on results incl. tables for details on results.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 2000 and 1000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of P1 females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
- At 1000 ppm, no effects were noted on parental fertility as assessed by normal mating, gestation and delivery.
- At 250 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery and development of their progeny.
As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 250 ppm test substance in the diet, corresponding to 16-25 and 24-31 mg/kg bw/day, should be regarded as NOAEL for P0 and P1generation.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: equivalent to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects on mating behaviour, fertility and gestation of each generation
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth was reduced. 

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly reduced pup body weight was observed. Pup weight gain was also slightly lower during lactation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight of the progeny, no other effects were noted on their development.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
See under Any other information on results incl. tables for details on results.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
ca. 250 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: a marginally to slightly lower mean food consumption and body weight gain and liver weights
Remarks on result:
other: corresponding to 24-31 mg/kg bw/day (12-15.5 mg a.i./kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mean age of preputial separation and vaginal opening was delayed due to reduced food intake
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, the litter size at birth were reduced (as a consequence of lower number of implantation sites of F1 parent females).
- At 1000 ppm, no effects were seen in F2 offspring regarding pup survival until weaning.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, slightly lower pup body weight was observed. The pup weight gain was slightly reduced during lactation, 
- At 1000 ppm, no effects were seen regarding pup development.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 2000 ppm, lower spleen weights were noted.
- At 1000 ppm, except for a marginally lower spleen weight, no other effects were noted on their development.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 2000 ppm, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.
At 1000 ppm, no effects were seen regarding pup development and after sacrifice at weaning.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general / developmental toxicity
Generation:
F2
Effect level:
ca. 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality
other: based on lower pup weights, lower pup weight gain during lactation, and upon necropsy dilatation of the cecum with feces was observed in 4/25 males and 2/25 females
Remarks on result:
other: corresponding to 96-123 mg/kg bw/day (48-61.5 mg a.i./kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

The mean achieved dosages of the test substance for the dose-levels of 250, 1000 and 2000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/day, respectively,
- females:

during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/day, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/day, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/day, respectively.

The actual intake of test substance for both males and females given 250, 1000 and 2000 ppm throughout the study is approximately 16-25, 61-101 and 123-208 mg/kg bw/day, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/day for the F1 generation.

Details on results:

Parent males (F0):

No effects:mortality, clinical signs, mating and fertility indices, seminology parameters, macroscopic post-mortem examination and histopathology.

 

Body weight:

 

During the first week of dosing, the body weight gains were slightly low at the low and mid dose-levels (-13% and -11%, p<0.01, p<0.05, respectively) and moderately low at the high dose-level (days 1 to 8, -24%, p<0.001). This effect on body weight gain was observed over all the dosing period (days 1 to 106, - 11%, p<0.01; -8%, non-statistically significant and -20%, p<0.001, at 250, 1000 and 2000 ppm, respectively).

At all dose-levels, when compared to controls, body weights were marginally lower for the low and mid dose-levels (between -5 and -7%, p<0.01) and slightly lower for the high dose-level during most of the dosing period (-13%, p<0.001).

 

Food consumption:the food consumption during the premating period was marginally low at 250 and 1000 ppm reaching statistical significance during the first weeks of dosing (weeks 1, 3 and 4) and sporadically during 5 or 7 weeks (between -7% and -10%, p< 0.01, p<0.001). This effect was more marked at 2000 ppm and was statistically significant during the whole dosing period (between -7 and -14%, p<0.01, p<0.001).

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in the males given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights and the absolute and body weight-related differences were dose-related in the males. These differences were considered to be due to the treatment with the test substance, C12-16 ADBAC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

 

Parent females F0

No effects:mortality, clinical signs, mating and fertility indices, estrous cycle, pregnancy and parturition data (implantation, fecundity, post-implantation, and neo-natal losses, gestation and delivery parameters) and histopathology.

Body weight:The body weight gain was unaffected by treatment at 250 and 1000 ppm. A moderately, statistically significantly, low body weight gain was observed at 2000 ppm during the first and the fourth week of premating (-31%, p<0.001 and -38%, p<0.01, respectively) and over all pregnancy (-13%, p<0.01) while it increased during the lactation period (24 g vs. 0 g, p<0.001). At this latter dose-level, body weights were lower during most of the dosing period (ranging between -4 and -9%, p<0.05, p<0.01) but returned to values comparable to controls at the end of the lactation period (316 g vs. 325 g in controls).

Food consumption:

The food consumption was unaffected by the treatment at 250 and 1000 ppm while it was slightly reduced at 2000 ppm, reaching statistical significance during the first week of dosing (-11%, p<0.001), during 2 weeks of the premating period (days 22 to 29 and days 64 to 71, -5% p<0.01) and during 2 weeks of the pregnancy period (days 0 to 7 p.c., -4%, p<0.05 and days 7 to 14,

-8%, p<0.001).

During the first week of the lactation period, group mean food consumption was slightly increased in the group given 2000 ppm and values were thereafter comparable to controls until the end of lactation.

 

Organ weights (expressed in %)

Lower absolute and body weight-related liver weights in females given 1000 or 2000 ppm were statistically significant. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were considered to be due to the treatment with the test item BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study.

Macroscopic post-mortem examination

Some segments of the intestinal tract were distended with faeces at necropsy with higher incidence in the females given 2000 ppm, compared with their respective controls.

Although no microscopic finding could be correlated, considering the higher incidence of this finding in the test item-treated females compared with their respective controls, distension of intestinal tract was considered to be related to treatment with the test item. This included:

-         ileum in 1/25 females given 1000 ppm and 5/25 females given 2000 ppm vs. none control female,

-         coecum in 1/25 females given 250 ppm, 8/25 given 1000 ppm and 12/25 females given 2000 ppm vs. 2/25 control females,

-         colon in 1/25 females given 1000 ppm and 2/25 females given 2000 ppm vs. none control female.

F1 males

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, seminology parameters and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was slightly lower at 2000 ppm (-13%, p<0.001) while it was minimally, but not statistically significantly,

affected at 1000 ppm (-5%) and was similar to controls at 250 ppm. During the whole dosing period, the body weight was generally slightly (for the low and mid-dose levels) to moderately (for the high dose-level), but statistically significantly, lower. The body weight gain was statistically significantly lower during the two first weeks of dosing at all dose-levels and remained markedly lower over all the dosing period at the high dose-level (days 1 to 120, - 18%,p<0.001).

Food consumption:

In the males, group mean food consumption was slightly to moderately lower at all dose-levels (ranging between -7 and -21%, p<0.05, p<0.01, p<0.001) reaching statistical significance during many periods at the low and mid dose- levels and during all recorded periods at the high dose-level (p<0.01, p<0.001).

Sexual development

The mean age at preputial separation (cleavage of the balanopreputial gland) was unaffected by the treatment at 250 and 1000 ppm (i.e occurred at 49.6 and

50.1 day old, respectively vs. 47.8 day old in controls) while, when compared to the male control group, a significant difference was observed in males given 2000 ppm (52.80 ± 6.02 vs. 47.76 ± 2.62 days, p<0.0019). It was not statistically significant when body weight was considered as a covariate, which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the males given 250, 1000 or 2000 ppm were observed, with statistical significance. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The coecum was distended with faeces in 1/25 males given 1000 ppm and in 4/25 males given 2000 ppm.

Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F1 females

No effects:mortality, clinical signs, neurobehavioral tests, mating and fertility indices, oestrous cycle, pregnancy and parturition data (with the exception of number of implantation sites and number of live foetuses), and histopathology.

Body weight:On day 1 (post-natal day 22), the body weight was statistically significantly lower at 1000 and 2000 ppm (-6%, p<0.005 and -13%, p<0.001, respectively) and remained lower over all the premating and pregnancy periods until the second week of lactation. The body weight gain was marginally reduced (-9%, not statistically significant and -15% p<0.001, for the low and mid-dose-levels, respectively) to markedly reduced (-41%, p<0.001 for the high dose-level) during the first week of premating and over the pregnancy periods (-10%, -11%, -19%, respectively, p<0.01 or p<0.001) while it was increased when compared to controls during the lactation period.

Food consumption:

In the females, group mean food consumption was unaffected by the treatment at the low and mid dose-groups while it was lower at 2000 ppm during premating and pregnancy, reaching statistical significance during most of the recorded periods (ranging between -9 and -14%, p<0.01, p<0.001). No effect was noted during the lactation period.

Sexual development

The mean age for vaginal opening was unaffected by the treatment, i.e. occurred on days 34.1, 33.7, 35.4 for pups from the control, 250 and 1000 ppm groups, respectively while it was delayed for 4 days at 2000 ppm.

When compared to the female control group, a significant difference was observed in females given 2000 ppm (38.40 ± 5.16 vs. 34.12 ± 2.19 in controls p<0.0001), however it was not statistically significant when body weight was considered as a covariate which suggests that this effect was the consequence of a lower body weight rather than a direct effect on sexual development.

Furthermore, animals that had values outside the range of individual control values had the lowest weight at sexual maturity which confirm that this effect was the consequence of reduced body weight and not a direct effect on sexual development.

Pregnancy and parturition data

The statistically significantly lower number of implantation sites and the lower number of live foetuses (-13%, p<0.001 for both parameters) in females given 2000 ppm when compared to controls, were considered to be treatment- related, although these values were within the range of historical control data.

Organ weights (expressed in %)

Lower absolute and/or body weight-related liver weights in the females given 250, 1000 or 2000 ppm were observed, with statistical significance and dose- relationship in the females. In addition, the absolute value of the differences observed was of higher magnitude than that observed for the differences in body weights. These differences were therefore considered to be due to treatment with the test item, BKC. It is worth mentioning that similar differences in liver weight without histomorphological changes were observed in the previous 90-day study and similar differences in liver weights were also noted in the F0 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

 

F2 males

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Macroscopic post-mortem examination

The cecum was distended with feces in 2/25 females given 2000 ppm. Although no microscopic finding could be correlated (at least for the macroscopic abnormalities which were examined), considering the previous observations made in the intestinal tract in the females of the F0 generation, this finding reported in a few test item-treated animals of both sexes, was considered to be most probably related to treatment with the test item.

F2 females

No effects:viability and lactation indices, clinical signs, gross external abnormalities, assessment of reflex and physical development (surface righting, cliff avoidance and air righting) and macroscopic post-mortem examination.

Body weight:No effects on body weight or body weight gain were noted at 250 and 1000 ppm while slightly lower body weight gain was observed at 2000 ppm between day 4 and day 21 post-partum (-8%, p<0.05).

Although this difference in body weight gain was slight, it was considered to be possibly related to the treatment.

Organ weights (expressed in %)

Considering that the differences in absolute and body weight-related weights of the spleen of the animals given 1000 or 2000 ppm were dose-related in the two sexes and that the absolute value of the difference in the absolute spleen weight was marginally higher than that observed for the difference in body weight at necropsy, a relationship to treatment could not be excluded, as similar changes were observed in the spleen weights of the pups of the F1 generation.

Offspring of F0 parents (F1 pups)

No effects:viability and lactation indices, clinical signs and gross external abnormalities, assessment of reflex development and macroscopic post-mortem examination.

Body weight:At birth, pup body weights were unaffected by the treatment at all dose-levels. During the lactation period, pup body weight and body weight gain were not affected at 250 ppm.

At 1000 and 2000 ppm, pup body weight gains were statistically significantly lower during the last week of lactation (-9%, p<0.05 and -11%, p<0.01) while body weight was statistically significantly lower on day 21 post-partum for the high-dose only (-7%, p<0.05). This effect was considered to be most probably related to pup food intake (which normally started during the last week of lactation) and was therefore considered to be related to the treatment.

Organ weights (pups sacrificed at weaning)

Dose-related lower absolute and body weight-related spleen weights were observed in the male and female pups given 1000 or 2000 ppm. This achieved statistical significance for the absolute weight for the animals given 2000 ppm. Almost all the individual weights were within the control range for the test item-treated animals; however they were gathered at the lower limit of the range, especially at the high dose-level.

For the differences in spleen weights, a relationship to treatment with the test item cannot be ruled out, as the same trend was observed in the pups of the F1 generation. However, they might be stress-related, considering the young age of the animals and/or the consequence, at least in part, of the decrease in body weight.

 

Results for F0 and F1 generation summarised according to dose groups:

The following results were obtained:

F0 generation (during premating, mating, pregnancy and lactation until day 21 post-partum of F0 parents and post-natal day 21 for their progeny)

Group 4: 2000 ppm

-        no treatment related clinical signs or mortality were noted,

-        for the parent males, over all the dosing period, statistically significantly low food consumption (between -7 and -14%, p<0.01 and p<0.001), low body weights (-6 and -13%, p<0.001) and reduced body weight gains (- 20%, p<0.001) were noted,

-        for the parent females, the food consumption was reduced during the first week of the premating period (-11%, p<0.001) and during the 2 first weeks of pregnancy (-9% and -8%, respectively). This was associated with lower body weight gain during the first week of the premating period (-31%, p<0.001), during the pregnancy period (-13%, p<0.01) and lower body weights over all the premating and pregnancy periods (between -4% and -9%, p<0.05, p<0.01). The food consumption and the body weight gains were slightly increased during the first week of lactation,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters) or on the progress of pregnancy or delivery,

-        no effect on pup survival (F1 generation pups) while reduced pup body weight gain was noted during the last week of lactation (-11%, p<0.01),

-        necropsy of parents revealed dilatation of ileum in 5/25 females, dilatation of cecum in 12/25 females and of the colon in 2/25 females,

-        lower absolute and body weight-related liver weights in parental males (respectively, -23%, -11%, p<0.01) and females (respectively, -16 % and

-        11%, p<0.01) and lower absolute and relative spleen weights of their progeny (respectively, -16%, p<0.05 and -9% for the males, -14%, p<0.05 and -8% for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

 

Group 2 and 3: 250 and 1000 ppm

-        no treatment related deaths or clinical signs were observed,

-        for the parent males, the food consumption was marginally low during the first week of dosing and sporadically during 4 or 5 weeks (between -7% and -10%, p<0.01, p<0.001) and was associated with a slightly low body weight gain during the first week of dosing (-13% and -11%, p<0.01, p<0.05, respectively) and over all the dosing period (-11%, p<0.05; -8%, respectively at both dose-levels),

-        for the parent females, the food consumption and body weight gains were unaffected by the treatment,

-        no effect on mating, male and female fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on progeny survival from delivery until weaning (F1 generation pups),

-        necropsy of parental males and females given 1000 ppm revealed dilatation of the cecum with feces in 8/25 females and dilatation of the ileum and the colon in 1/25 females,

-        at 1000 ppm, there were lower absolute and body weight-related liver weights of parental males (respectively, -12%, p<0.05 and -7%, p<0.05) and females (-14% and -11%, p<0.01) and marginal, non-statistically significant, lower absolute and body weight-related progeny spleen weights (-7% and -4% for the males and -9% and -6% for the females),

-        at 250 ppm, no effect was noted on parents or progeny‟ organ weights,

-        no histopathological findings were noted at microscopic examination of  the different parental organs (including reproductive organs) at either dose-level.

 

F1 generation (from post-natal day 22, during mating, pregnancy and lactation until day 21 post-partum)

Group 4: 2000 ppm

-        no treatment-related clinical signs or mortality in parental males or females,

-        for the males, the food consumption was lower during the whole dosing period (between -12 and -21%, p<0.0001) and the body weight and body weight gain were moderately lower over all the dosing period (-18%, p<0.001),

-        for the females, reduced food consumption was noted during the premating and pregnancy periods (ranging between -9% and -14%, p<0.01 and p<0.001). This was associated with lower body weight from the first day of the premating period until the second week of lactation and lower body weight gain during the first week of premating (-41%, p<0.001) and over all the premating and pregnancyperiods,

-        the mean age of preputial separation and vaginal opening was delayed   for approximately 5 and 4 days, respectively (52.8 vs. 47.76 day old for preputial separation and 38.4 vs. 34.12 day old for vaginal opening) as a consequence of lower body weight, while no effect was noted on their locomotor activity or on neurobehavioralparameters,

-        no effects were noted on mating or parental fertility (including number of pregnant females with liveborn, estrous cycle and seminology parameters), however, the number of implantation sites and the litter size at birth were reduced (13 vs. 14.9 and 12.4 vs. 14.2, p<0.01, respectively),

-        the pup weight gain of the progeny of the F1 generation (F2 generation pups) was slightly lower during the lactation period (-8%,p<0.05),

-        necropsy of the F1 generation revealed dilatation of the cecum with feces in 4/25 males and 2/25females,

-        lower absolute and body weight-related liver weights of parental males (respectively, -28% and -12%, p<0.01) and females (respectively, -25% and -18%, p<0.01) and decreased absolute and relative spleen weights of their progeny (-23%, p<0.01 and -10% for the males, -26% and -14%, p<0.01 for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductiveorgans).

 

Group 3: 1000 ppm

-        no treatment-related clinical signs in parental males andfemales,

-        for the males, the food consumption was statistically significantly lower during many dosing periods (between -6% and -11%, p<0.05, p<0.01, p<0.001) and was associated with a slightly lower body weight and body weight gain over all the dosing period (-7%),

-        for the females, the body weight and food consumption were slightly lower during the first week of premating and during pregnancy (body weight: -15%, p<0.001 and -11%, p<0.01, food consumption: between -9 and -14%, p<0.01,p<0.001),

-        no effect on mating, parental fertility (including estrous cycle and seminology parameters), gestation, fecundity ordelivery,

-        no effect on pup development and progeny survival from delivery until weaning,

-        no effect on locomotor activity and neurobehavioural parameters of the progeny,

-        necropsy of the parents revealed dilatation of coecum with faeces in 1/25 males,

-        lower absolute and body weight-related liver weights of parental males (respectively, -13%, p<0.05 and -6%, not statistically significant) and females (respectively, -17% and -14%, p<0.01) and marginally lower absolute and body weight-related spleen weight of their progeny (-3% for the males and -11 and -10%, respectively for the females),

-        no histopathological findings were noted at microscopic examination of  the different organs (including reproductive organs).

Group 2: 250 ppm

-        no effects at clinical examination of the male and female parents,

-        for the males, body weight and body weight gain were slightly, but statistically significantly, lower during most of the dosing period (-9%, p<0.05) while the food consumption was transiently lower,

-        for the females, the body weight gain was slightly lower during pregnancy (days 0 to 20 p.c., -10%, p<0.01) while no effect was noted on food consumption,

-        no effect on mating, male and female parental fertility (including estrous cycle and seminology parameters), gestation, fecundity or delivery,

-        no effect on sexual development or on neurobehavioral parameters,

-        no effect on the development of the progeny from delivery until weaning,

-        no macroscopic findings in parental males and females or their progeny,

-        lower absolute and body weight-related liver weights of parental males (respectively, -14%, p<0.05 and -6%, not statistically significant) and females (respectively, -14% and -12%, p<0.01) but no effect was observed on organ weights of the progeny,

-        no microscopic findings for male and female parents.

Conclusions:
Based on the results of the read across study, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively].
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, C12 -16 ADBAC (49.9% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1000 and 4000 ppm (corresponding to 0, 250, 1000 and 2000 ppm a.i. and equivalent to 0, 16-25, 61-101 and 123- 208 mg a.i./kg bw/day, respectively for the F0 generation and 24-31, 96-123 and 202-252 mg a.i./kg bw/day for the F1 generation respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 2000 ppm, a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilatation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 1000 ppm, P0 (males) and P1 (both sexes) showed a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 250 ppm (corresponding to approximately 16-25 mg a.i./kg bw/day for F0 males and females and 24-31 mg a.i./kg bw/day for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.The lower food intake, which was most pronounced in period of start of intake as well in F0 as in F1 animals, indicated that the intake was influenced by palatability of the compound and not by a toxic mechanism. At 250 ppm, the small effects on body weight, body weight changes and absolute and relative organ weights were considered to be resulting from reduced food intake. Similar was the case for the 1000 ppm group. As this was not considered to be an adverse toxic effect from exposure to the read across substance, 250 ppm was considered to be the NOAEL for parental systemic toxicity. The NOEL for mating behaviour, fertility and gestation of each generation was considered to be 1000 ppm. The marginally lower absolute and body weight-related spleen weight of the F2 progeny at 1000 ppm was also considered to be indirectly caused by the lower food intake of F1 parents, and therefore was not considered to be an adverse toxic effect from exposure to the read across substance. Therefore, the NOAEL for development, growth and survival of each progeny was considered to be at 1000 ppm. Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively] (Foulon, 2008). Based on the results of the read across study, similar NOAELs can be expected for the test substance.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: Six weeks
- Weight at study initiation: 212.2-213.4 g (males); 148.3-150.2 g (females)
- Housing: Individually in stainless steel, wire mesh cages (22.5x15.5x18.0 cm; mating cages 22.5x31.0x18.0 cm)
- Diet: Certified Ground Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack.
- Acclimation period: Two weeks

Environmental conditions
- Temperature: 66-73°F
- Humidity: 40-60%
Route of administration:
oral: feed
Vehicle:
other: Certified Ground Rodent Chow # 5002
Details on exposure:
Diet preparation: A concentrated premix was prepared by direct addition of the test substance to ground chow and mixing for approximately an hour. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared weekly.
- Mixing appropriate amounts with (Type of food): Certified Ground Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polyethylene containers at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Mating procedure: After pre-breeding exposure the animals were mated on the basis of one male to one female selected randomly within each dose group for a period of 21d to produce F1 generation.
- Proof of pregnancy: Copulation plug and/or vaginal sperm as Day 0 of gestation.
- After the first 7d of the mating period females of unsuccessfully mated pairs were placed with males of other unmated pairs within the same dose group; after an additional 7d, unsuccessfully mated pairs were similarly exchanged again for a period of 7d or until successful mating had occurred, whichever came first, allowing for a total of 21d to mate.
- After successful mating each pregnant female was caged: Pregnant females were housed individually. On Day 20 of gestation each pregnant female was transferred to a shoe-box cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance using high pressure liquid chromatography.
- Homogeneity study indicated that the distribution of the test substance in the test diet was uniform.
- Stability study indicated that the dosed feed was stable for at least 14d when stored in open glass feed jars at room temperature. Dosed feed was stable for at least 21d when stored in closed polyethylene containers at room temperature.
- Concentration verification analyses of the dosed feed indicated that the mean concentrations of the test substance in the diet for the 300, 1000 and 2000 ppm dosage levels were 95.3-109.0% of nominal for 300 ppm, 95.6-107.9% of nominal for 1000 ppm and 94.7-108.0% of nominal for 2000 ppm.
Duration of treatment / exposure:
P0 generation: 19 weeks (from 1st prebreed dose to last F0 sacrifice)
F1 generation: 25 weeks (from 1st F1 wean to last F1 sacrifice)
F2 generation: Until weaning
Frequency of treatment:
Daily
Details on study schedule:
- P1 parental animals not mated until 17-18 weeks after selection from the F1 litters.
- Selection of parents from F1 generation when pups were 28d old.
- Number of P1 generation animals selected: 28 males and 28 females

Remarks:
Doses / Concentrations:
0, 300, 1000 or 2000 ppm test substance (i.e., equivalent to 0, 16-31, 51-102, and 100-188 mg/kg bw/day (males) and 0, 21-32, 67-106 and 139-198 mg/kg bw/day (females)).
Basis: nominal in diet
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: Animals were randomly distributed based on body weight
- Animal identification: By ear tags
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Once daily for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreed and mating for both sexes. For females on gestational Day 0, 6, 15, and 20 and on postnatal Day 0, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Weekly during prebreed for both sexes. For females in 3- or 4-day intervals throughout gestation and to postnatal Day 14.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Excess pups were subjected to detailed external examination and then sacrificed.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: Live/Still births on the day of birth (postnatal Day 0), survival indices at Days 0, 4, 7 and 14 after birth and weaning, weight on postnatal Day 1, 4, 7, 14, and at weaning (Day 21), and physical abnormalities for all pups at birth and throughout the pre-weaning period.

GROSS EXAMINATION OF DEAD PUPS: The thoracic and abdominal organs from pups which died after Day 4 were preserved for subsequent histopathological examination.
Postmortem examinations (parental animals):
SACRIFICE: Yes
- How many animals: All animals
- Necropsy method: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.

GROSS NECROPSY: Yes
- How many animals: All animals sacrificed in F0 and F1 parental animals
- Gross necropsy consisted of: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate.

HISTOPATHOLOGY : Yes
- How many animals: All animals of control and high dose groups sacrificed in P0 and P1 parental animals
- Tissues evaluated: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate and any tissues or organs showing gross alterations from the low and mid dose groups
Postmortem examinations (offspring):
SACRIFICE
- The F1 offsprings not selected as parental animals and all F2 offsprings were sacrificed at 7d of age.

GROSS NECROPSY
- Examination for gross lesions was performed on any pup appearing abnormal or dying on test and for ten randomly selected F1 and F2 weanlings/sex/group.

Statistics:
- The results of the quantitative continuous variables (e.g., body weights, food consumption, etc.) were compared between the three treatment groups and one control group using Levene’s test for equal variances, analysis of variance and (pooled or separate variance) t-test.
- Non-parametric data were statistically evaluated using the Kruskall-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate.
- Frequency data were compared using the Fisher’s exact test.
Reproductive indices:
Mating index, fertility index and gestational index were determined.
Offspring viability indices:
Live birth index, 4-d survival index, 7-d survival index, 14-d survival index, 21-d survival index and lactation index

Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were observed.



Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- During the 10-week pre-breed exposure, F0 males exhibited no reduction in body weight. During the same period, F0 females at 2000 ppm exhibited reduction in body weight during Weeks 5, 6, 9 and 10 of pre-breed treatment. Body weight gain was also reduced at 2000 ppm for one week (Week 8-9) during the pre-breed period.
- On lactation Day 21 mean body weight of F0 dams at 2000 ppm exhibited a significant increase. Increased lactation body weight gain was observed at 2000 ppm throughout lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption in the F0 females at 2000 ppm was reduced for the first four exposure weeks. Food consumption in F0 males was significantly reduced at 2000 ppm for the first week of treatment only.
- At F0 breed to produce F1 litters, food consumption during gestation and lactation was unaltered by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
No treatment-related effects on any reproductive parameters were observed at any dose; NOEL (parental) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
other: body weight and food consumption
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 2000 ppm, only slight reduction were observed in males and females
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity in the F1 or F2 animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effects of treatment on postnatal deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The F1 litters exhibited reduced body weight per litter on postnatal Days 21 and 28 at 2000 ppm. F1 pup body weight gain was reduced during lactation Days 14-21 and 21-28.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
NOEL (F1and F2 offspring) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity in the F1 or F2 animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effects of treatment on postnatal deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 pup weights per litter were reduced at 2000 ppm on postnatal Day 28. Pup weight gain was also reduced at 2000 ppm during lactation Days 14-21 and for Days 21-28.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy.
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
NOEL (F1and F2 offspring) = 1000 ppm
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

For result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:
Based on the results of the read across study, the rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females.
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) according to US EPA OPP 83-4, in compliance with GLP. The read across substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm read across substance (equivalent to average doses of 0, 16-31, 51-102, and 100-188 mg/kg bw/day for males and 0, 21-32, 67-106 and 139-198 mg/kg bw/day in females) in the diet. Following a 10-week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3-week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, twenty-eight (28) F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the read across substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and F1 animals were necropsied and examined for gross lesions. Selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. Remaining non-selected F1 and F2 pups at weaning were euthanised and discarded. Reduced body weights in F0 males (but no F0 males or F1 males and females), reduced weight gain in F0 males and females and F1 males (but not F1 females) and reduced food consumption in F0 males and females and F1 males (but not F1 females) during the ten-week pre-breed exposures was observed. With the exception of body weights and food consumption in F0 females, reductions in the pre-breed parameters appeared transitory, disappearing after one or two weeks. Reproductive parameters were not affected in either of the two breeds (F1 or F2). At initiation of the gestational period, body weights of the F0 (but not F1) females at 2000 pp, were reduced; weight gains through-out gestation for both breeds were normal. Reduced gestations food consumption was observed in F1 females only. F1 litters at 2000 ppm exhibited reduced body weights at weaning; both F1 and F2 generations of pups exhibited reduced body weights on Day 28 postpartum, one week subsequent to weaning. Body weight gains in both F1 and F2 litters were reduced for corresponding time intervals (lactational days 14-21 and 21-28) as well. The reduction in pup body weights (and weight gain) at 2000 ppm was considered to be treatment related as it corresponded to the time when the pups began to rely solely on the read across substance diet as their source of nutrition. While the statistical reductions were observed only for female pup body weight gains on lactation days 21-28 in F1 litters and for male pups on days 14-21 in F2 litters, body weight gain reductions were apparent in both sexes of pups during these time periods. There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy. Under the conditions of the study, it can be stated that, dietary administration of the read across substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Also, there was no increased risk to the offspring in the absence of indications of maternal toxicity. The rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females (Neeper-Bradley, 1990). Based on the results of the read across study, similar NOAELs can be expected for the test substance.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
16 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Guideline compliant read across studies
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Study 1: A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, C12 -16 ADBAC (49.9% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1000 and 4000 ppm (corresponding to 0, 250, 1000 and 2000 ppm a.i. and equivalent to 0, 16-25, 61-101 and 123- 208 mg a.i./kg bw/day, respectively for the F0 generation and 24-31, 96-123 and 202-252 mg a.i./kg bw/day for the F1 generation respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 2000 ppm, a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilatation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 1000 ppm, P0 (males) and P1 (both sexes) showed a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 250 ppm (corresponding to approximately 16-25 mg a.i./kg bw/day for F0 males and females and 24-31 mg a.i./kg bw/day for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.The lower food intake, which was most pronounced in period of start of intake as well in F0 as in F1 animals, indicated that the intake was influenced by palatability of the compound and not by a toxic mechanism. At 250 ppm, the small effects on body weight, body weight changes and absolute and relative organ weights were considered to be resulting from reduced food intake. Similar was the case for the 1000 ppm group. As this was not considered to be an adverse toxic effect from exposure to the read across substance, 250 ppm was considered to be the NOAEL for parental systemic toxicity. The NOEL for mating behaviour, fertility and gestation of each generation was considered to be 1000 ppm. The marginally lower absolute and body weight-related spleen weight of the F2 progeny at 1000 ppm was also considered to be indirectly caused by the lower food intake of F1 parents, and therefore was not considered to be an adverse toxic effect from exposure to the read across substance. Therefore, the NOAEL for development, growth and survival of each progeny was considered to be at 1000 ppm. Under the study conditions, the rat NOAEL (systemic toxicity) for both parental generation (F0 and F1) was considered to be at 250 ppm [equivalent to 16-25 mg/kg bw/day (or 8-12.5 mg a.i./kg bw/day) for F0 and 24-31 mg/kg bw/day (or 12-15.5 mg a.i./kg bw/day) for F1 generation, respectively]. The rat NOEL (reproductive toxicity) and NOAEL (developmental toxicity) was considered to be at 1000 ppm [equivalent to 61-101 mg/kg bw/day (or 30.5-50.5 mg a.i./kg bw/day) for F0 and 96-123 mg/kg bw/day (or 48-61.5 mg a.i./kg bw/day) for F1 generation, respectively] (Foulon, 2008).

Study 2: A two-generation study was conducted to determine the toxicity to reproduction of the read across substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) according to US EPA OPP 83-4, in compliance with GLP. The read across substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm read across substance (equivalent to average doses of 0, 16-31, 51-102, and 100-188 mg/kg bw/day for males and 0, 21-32, 67-106 and 139-198 mg/kg bw/day in females) in the diet. Following a 10-week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3-week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, twenty-eight (28) F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the read across substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and F1 animals were necropsied and examined for gross lesions. Selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. Remaining non-selected F1 and F2 pups at weaning were euthanised and discarded. Reduced body weights in F0 males (but no F0 males or F1 males and females), reduced weight gain in F0 males and females and F1 males (but not F1 females) and reduced food consumption in F0 males and females and F1 males (but not F1 females) during the ten-week pre-breed exposures was observed. With the exception of body weights and food consumption in F0 females, reductions in the pre-breed parameters appeared transitory, disappearing after one or two weeks. Reproductive parameters were not affected in either of the two breeds (F1 or F2). At initiation of the gestational period, body weights of the F0 (but not F1) females at 2000 pp, were reduced; weight gains through-out gestation for both breeds were normal. Reduced gestations food consumption was observed in F1 females only. F1 litters at 2000 ppm exhibited reduced body weights at weaning; both F1 and F2 generations of pups exhibited reduced body weights on Day 28 postpartum, one week subsequent to weaning. Body weight gains in both F1 and F2 litters were reduced for corresponding time intervals (lactational days 14-21 and 21-28) as well. The reduction in pup body weights (and weight gain) at 2000 ppm was considered to be treatment related as it corresponded to the time when the pups began to rely solely on the read across substance diet as their source of nutrition. While the statistical reductions were observed only for female pup body weight gains on lactation days 21-28 in F1 litters and for male pups on days 14-21 in F2 litters, body weight gain reductions were apparent in both sexes of pups during these time periods. There were no treatment-related observations or histopathological findings in either the F0 or F1 adult animals at any dose. There were no treatment-related findings in the F1 or F2 pups that died during lactation or randomly selected F1and F2 pups at necropsy. Under the conditions of the study, it can be stated that, dietary administration of the read across substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Also, there was no increased risk to the offspring in the absence of indications of maternal toxicity. The rat NOAEL (systemic toxicity) for both parental generation (P0 and F1) and offspring (F1 and F2) was considered to be at 1000 ppm (in diet), i.e., equivalent to 51-102 mg/kg bw/day (or 41-83 mg a.i./kg bw/day) in males and 67-106 mg/kg bw/day (or 54-86 mg a.i./kg bw/day) in females, respectively. The rat NOEL (reproductive toxicity) although not specified by the authors in the study report, can be considered to be at the highest tested dose 2000 ppm (in diet), i.e., equivalent to 100-188 mg/kg bw/day (or 81-152 mg a.i./kg bw/day) in males and 139-198 mg/kg bw/day (or 113-161 mg a.i./kg bw/day) in females (Neeper-Bradley, 1990).

In line with the biocides assessment report, the NOAELs from the 2-generation reproductive toxicity studies in rats have not been considered further for systemic risk assessment. The biocides assessment reports available from RMS Italy on C12-16 ADBAC and Coco TMAC, overall concluded similarly that these substances do not indicate a concern for reproduction toxicity. However, the corrected dietary doses were slightly different in the C12-16 ADBAC biocides assessment report, where the NOAELs for parental and offspring toxicity were both reported be >50 and >30 mg/kg bw/day and the NOAELs for effects on fertility was reported to be >100 and >52 mg/kg bw/day for the Foulon, 2008 and Neeper-Bradley, 1990 studies respectively (ECHA biocides assessment report, 2015, 2016). The assessment report on Coco TMAC, which contained only the Foulon 2008 study however corresponded to the NOAELs summarized in this dossier. Nevertheless, due to the absence of specific reproductive toxicity and the fact that the observed effects in parents relate to general toxicity which are secondary to local effects, the parental NOAELs (16 mg/kg bw/day) were not considered to be relevant to the determination of a systemic DNELs.

Effects on developmental toxicity

Description of key information

The available oral and dermal pre-natal development toxicity study with the test and read across substances in rats and rabbits, indicate no concern for development toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 March 2020 to 07 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Animal supply and acclimatisation
A total of 120 Crl:CD Sprague Dawley (SD) virgin female rats, 9 weeks old (200-225 g) were supplied and received from Charles River Italia S.p.A., Calco (Lecco), Italy. A first batch of 100 females arrived on March 5, 2020 and a second batch of 20 females arrived on March 12, 2020. The male rats used for mating were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of 18 days for the first batch of animals and 11 days for the second batch was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal husbandry
The rats were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C 2°C and 55% 15%, respectively; actual conditions were monitored, recorded and the records retained. No deviations from these ranges were recorded during the study. There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5 38 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5 26.6 18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the mated females were housed individually in clear polysulfone cages meas- uring 42.5 26.6 18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. The certificate of analysis of the nesting material used was kept with study raw data. Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. The certificate of the diet used was kept with study raw data. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened water (by reverse osmosis)
Details on exposure:
All animals were administered the test substance by oral gavage during the gestation period, starting from Day 3 through Day 19 post coitum at the doses of 0, 18.75, 37.5, 75 mg/kg bw/day (25 animals in each group).

Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation (SOP ANI/105) to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed individually. The cages were identified by a label recording the study number, animal number and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in ERBC Study no. A3762 in the range from 1 to 10 mg/mL. The proposed preparation procedure for the test substance was checked in the range from 1 to 10 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A3762 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (85-115%) and homogeneity (CV < 10%). In the same study (ERBC Study no. A3762) and in the same range of concentration, the stability of the preparations was verified and the final results were as follows: (a) at 28 hours at room temperature (b) at 10 day stability at 2-8°C. Samples of the preparations prepared on Week 1 and Last week of treatment were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV <10% for homogeneity). The validated software used for this activity was Empower® 2 Build No. 2154.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum
Frequency of treatment:
Once a day
Remarks:
0, 18.75, 37.5, 75 mg/kg bw/day
The required amount of test substance was suspended in the vehicle. The preparations were made at up to 8- day intervals (concentrations of 1.875, 3.75 and 7.5 mg/mL). Concentrations were calculated and expressed in terms of test substance corrected for purity.
No. of animals per sex per dose:
Each group comprised 25 mated female rats
Control animals:
yes
Maternal examinations:
Throughout the study, all animals were checked for mortality early in the morning and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice. All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum. Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum. On Day 20 post coitum, blood samples for thyroid hormones determination (T3, T4 and TSH) (approximately 0.5 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA). As a part of the terminal sacrifice procedure, blood samples for haematological investigations were withdrawn from the abdominal vena cava, of all females, under isoflurane anaesthesia, and collected in tubes with EDTA anticoagulant.

All animals were killed by exanguination under isoflurane anaesthesia on Day 20 post coitum. All animals were subjected to necropsy, supervised by a pathologist. From all animals completing the scheduled test period, the thyroid was dissected free of fat, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to body weight was calculated for each animal. After dehydration and embedding in paraffin wax, sections of the thyroid tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes in all groups.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. In addition, the anogenital distance (AGD) was recorded in all male and females foetuses. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter were preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal (single staining) examination. Skeletal and fixed-visceral examinations were performed in all groups. In both cases, sex was confirmed by internal inspection of the gonads.

Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal- Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Group mean values for body weight and food consumption of pregnant females, gravid uterus weight, organ weight, absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), litter size, intra-uterine deaths, corpora lutea count, number of implantations, total implantation loss, pre- and post-implantation loss, sex ratio, anogenital distance, haematology and thyroid hormone determination were calculated. Data from non-pregnant animals were not included in group mean calculations of maternal body weight.

Sex ratios of the foetuses were calculated as the percentage of males. The anogenital distance (AGD) was calculated as normalized to the cube root of body weight measured on Day 20 post coitum. All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Historical control data:
The AGD values range of laboratory historical control data were 1.70 - 3.74 mm/g1/3 for males and 0.96 - 3.00 mm/g1/3 for females.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was the treatment-related clinical sign observed in all treated groups with a dose related incidence: 6 out 25 in Group 2 (18.75 mg/kg bw/day), 17 out of 25 in Group 3 (37.5 mg/kg bw/day) and 23 out of 25 in Group 4 (75 mg/kg bw/day). In Group 4 (75 mg/kg bw/day) this sign was accompanied by hunched posture in a few females. The onset of these signs was on Day 5 post coitum in Group 4 and on Day 7 post coitum in Groups 2 and 3. Minor signs, such as hairloss and damaged ear were sporadically recorded during the study and were considered as incidental.
Mortality:
no mortality observed
Description (incidence):
No animals died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease (up to -6%) in body weight was observed in females receiving 75 mg/kg bw/day (Group 4) on Days 9, 12 and 20 post coitum. Body weight gain was statistically significantly reduced in Group 4 relative to the control group on two occasions, Days 6 and 20 post coitum. However, values between Days 9 and 18 were comparable to control. Body weight and body weight gain in females receiving 18.75 mg/kg bw/day (Group 2) and 37.5 mg/kg bw/day (Group 3) were comparable to the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in food consumption was observed in females receiving 75 mg/kg/day (Group 4) starting from Day 6 post coitum (approximately -29%) through Day 15 post coitum (approximately -8%). No differences were recorded on Day 18 post coitum and again a slight decrease was recorded on Day 20 post coitum (approximately -20%). No differences in food consumption were noted in females of Group 3, compared to controls. The statistically significant decrease (approximately -7%) in food consumption noted in females of Group 2 on one occasion was not considered of toxicological relevance.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
An increase of polychromatophil erythrocytes was observed in the peripheral blood smears of a number of animals of all treated groups. This finding was dose-related in the incidence. Due to the dose-relation observed between groups, the relation with the test item cannot be definitively excluded, even if there were no indications of an effect of the test item on the bone marrow or other tissues/organs. Considering that: (a) no haematological findings were observed (e.g. anaemia) (b) no changes were recorded during the in-vivo phase or at post-mortem examination (c) no treatment-related effects were observed in previous studies (https://echa.europa.eu/it/registration-dossier/-/registered-dossier/14219/7/6/2), the increase of polychromatophil erythrocytes alone was considered to be not adverse.

In addition, monocytes group mean data were statistically significantly higher than controls in females dosed at 75 mg/kg/day (30%). Since the increase of monocytes is usually associated with chronic inflammation/necrosis and no related findings were recorded (e.g. lymphocytosis), and similar monocytes values were found in two control females, the above increase of monocytes was considered to be incidental.

No differences in haematology parameters were noted in females receiving 18.75 mg/kg/day (Group 2) and 37.5 mg/kg/day (Group 3), compared to controls.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No differences between control and treated females were recorded in thyroid hormone.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on the thyroid weight at any dose levels, compared to the control group.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in thyroid gland of females receiving Cetrimonium chloride at low, medium and high dose.
Details on results:
Statistically significant reductions in terminal body weight (approximately -6%) and gravid uterus weight (-11%), associated with reduction (-29%) in absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), were seen in females receiving 75 mg/kg/day (Group 4), compared to controls. No differences in these parameters were noted in females receiving 18.75 mg/kg/day (Group 2) and 37.5 mg/kg/day (Group 3), compared to controls.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No differences in the uterine deaths (early, late and total) were observed.
Details on maternal toxic effects:
No mortality occurred during the study. During the in-life phase, maternal toxicity was noted in females receiving 75 mg/kg bw/day, as indicated by the clinical signs and the reduction in the absolute weight gain. The slight and occasionally decrease of body weight, body weight gain,food consumption, terminal body weight, gravid uterus weight and mean foetal weight were not considered of toxicological relevance since all these findings were of low magnitude and did not affect the pregnancy and developmental outcomes thus, they were considered not adverse. At macroscopic observations, no relevant changes that could be considered treatment- related were recorded. Polychromasia detected at haematology investigation was considered to be not adverse, even though the relation with the test item cannot be excluded. No differences between control and treated females were recorded in thyroid hormones determination, thyroid weight and thyroid histopathology. No difference considered treatment related was noted in the mean values of the anogenital distance of foetuses of both sexes maternally exposed at all dose levels compared to the control group. On the basis of the above results, it can be concluded that the dosage of 75 mg/kg bw/day induced slight maternal toxicity with no adverse effect on pregnancy. The dosages of 37.5 mg/kg bw/day and 18.75 mg/kg bw/day were well tolerated. The NOAEL for maternal toxicity was considered to be 75 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 75 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: all parameters
Remarks on result:
other: only slight maternal toxicity with no adverse effect on pregnancy
Remarks:
the effects were either toxicologically not relevant (e.g., decrease of body weight, body weight gain, food consumption, terminal body weight, gravid uterus weight and mean foetal weight) due to their low magnitude of change
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease was noted in the mean foetal weight, for each sex (approximately -4 and -5% for females and males respectively), as well as for both sexes combined (approximately-4%). However, considering that the value were within the range of our historical control data and considering dering the limited magnitude of the difference, the finding was not considered to be toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 14 small foetuses (< 2.7 g) were detected, 9 out of 356 in the control group, 1 out of 322 in the low dose group, 1 out of 354 in the mid-group and 3 out of 346 in the high dose group. One foetus in the mid-dose group had alterations classified as malformations: imperforate anus and absence of tail (acaudia).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were detected in control and in treated groups without dose relationship, in terms of foetuses and litters affected, as reported in attached results tables in attached background material section of the IUCLID. The other alterations (anomalies and variations) were seen in treated and control foetuses with similar incidence and/or without dose relationship. The type and distribution of mentioned findings, both for major and minor alterations, were considered incidental and not treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were detected in mid- and high dose groups as reported in attached results tables in attached background material section of the IUCLID. A total of two foetuses showed malformations: heart ventricle enlarged extreme was observed in one foetus maternally exposed at 37.5 mg/kg bw/day (Group 3) and extremely enlarged ureter in association with kidney pelvic dilatation extreme in one foetus maternally exposed at 75 mg/kg bw/day (Group 4). These findings were considered incidental since they were seen in single foetuses from different litters and their incidence was lower when compared with laboratory historical control data. The other alterations (anomalies and variations) recorded were noted both in control and treated groups, with a quite similar incidence.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences in AGDwere noted in male foetuses of mid- and high dose groups (approximately + 0.8% and + 3%, respectively) and in female foetuses of low and mid- dose groups (approximately -4% and -2.7%, respectively), compared to the control. Considering that the AGD reflects the prenatal androgenic exposure, with values physiologically higher in males than in females, the decrease observed in female foetuses represents a trend towards feminisation (reduction in AGD of females) and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation). The same applies to the increase observed in males, that also represents a trend towards masculinisation (increase in AGD of males) and is not considered an adverse effect (i.e. decrease in AGD of males, suggesting feminisation). Thus, these differences were considered incidental and non-adverse effects.
Details on embryotoxic / teratogenic effects:
The slight decrease noted in the mean foetal weight, for each sex as well as for both sexes combined, was not considered toxicologically relevant, considering the limited entity of the differences compared to control. The alterations noted at external, skeletal and visceral examinations of foetuses were con- sidered incidental and not treatment-related since they were quantitatively similar between control and treated groups with an absence of doses-related response. Based on these results, the NOAEL for developmental toxicity was considered to be 75 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 75 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: all parameters
Remarks on result:
other: effects were either toxicologically not relevant (decrease in foetal weight) or incidental and not treatment related (skeletal and visceral examinations)
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Table 15: Skeletal examination of foetuses

Organ

Malformation

Groups (mg/kg/day)

 

 

1 (0)

2 (18.75)

3 (37.5)

4 (75)

Pelvic girdle

Pubis not ossified

4(2)

3(2)

-

-

Ischium not ossified

-

1(1)

-

-

Skull

Presphenoid not ossified

2(2)

3(2)

1(1)

4(2)

Squamosal abnormal shape

-

-

-

1(1)

Cleft palate

-

1(1)

-

-

Forelimbs

Radius, ulna and humerus misshaped

-

-

1(1)

-

Ribs

Fused

-

-

2(1)

-

Thoracic vertebrae

Haemivertebra

-

-

1(1)

 

Absence of thoracic arch

-

-

1(1)

 

 

 

Foetus (litter)

Table 16: Visceralexaminationoffoetuses

Organ

Malformation

Groups (mg/kg/day)

 

 

1 (0)

2 (18.75)

3 (37.5)

4 (75)

Heart

Ventricle enlarged extreme

-

-

1(1)

-

Kidney/Ureter

Pelvic dilatation extreme/Enlarged extreme

-

-

-

1(1)

Foetus (Litter)

For other result tables, kindly refer to the attached background material section of the IUCLID.

Conclusions:
Under the study conditions, the NOAEL for maternal and developmental toxicity was determined to be 75 mg/kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity potential of C16 TMAC (purity: 98.5%), using prenatal developmental toxicity oral gavage study in female Sprague Dawley rats, according to the OECD Guideline 414, in compliance with GLP. All animals were administered the test substance during the gestation period, starting from Day 3 through Day 19 post coitum at the doses of 0, 18.75, 37.5, 75 mg/kg bw/day (25 animals in each group). Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post-mortem examination. Blood collection for haematology and hormone determination, in association with determination of the thyroid weight from all females was performed on Day 20 post coitum. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities. No animals died during the study. A total of three females were found not pregnant at necropsy: one in the control group, one in the low dose group (18.75 mg/kg bw/day) and one in the mid-dose group (37.5 mg/kg bw/day). One low dose female had unilateral implantation in the left horn and was not pregnant in the right one. The number of females with live foetuses on Day 20 post-coitum was: 24 in the control, low and mid-dose groups and 25 in the high dose group (75 mg/kg bw/day). Piloerection was the treatment-related clinical sign observed in all treated groups with a dose-related incidence. In Group 4 this sign was accompanied by hunched posture in few females. Minor signs, such as hair-loss and damaged ear were sporadically recorded during the study and were considered incidental. The slight statistically significant decreases in body weight observed in females of Group 4 was not considered of toxicological relevance due to the limited magnitude of the change. These changes were considered related to treatment, but not adverse. Statistically significant reduction in food consumption was observed in females of Group 4 starting from Day 6 post coitum through Day 15 post coitum. No differences were recorded on Day 18 post coitum and again a slight decrease was recorded on Day 20 post coitum. This change was considered not relevant because there was a recovery between the start and the end of treatment. The polychromasia observed in all treated groups was considered to be not adverse, even though the relation with the test substance cannot be excluded. Monocytosis observed in females dosed at 75 mg/kg bw/day was considered to be incidental. The increase of polychromatic cells in the peripheral blood smear even when the reticulocyte count is normal could suggests the premature release of erythroid cells from a marrow that is not hyperactive. Considering that: (a) no haematological findings were observed (e.g. anaemia) (b) no changes were recorded during the in-vivo phase or at post-mortem examination (c) no treatment-related effects were observed in previous studies (https://echa.europa.eu/it/registration-dossier/-/registered-dossier/14219/7/6/2). There were no indications of an effect of the test substance on the bone marrow or other tissues/organs which could led to an increase of the polychromatic cells, therefore this finding was considered to be not adverse. Due to the dose-relation observed between groups, the relation with the test substance cannot be definitively excluded. In thyroid hormone determination, no differences between control and treated females were recorded. There was no effect on the thyroid weight at any dose levels, compared to the control group. Statistically significant reductions in terminal body weight, gravid uterus weight and absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), seen in females of Group 4, compared to controls were considered not adverse due to the limited magnitude of the change. No differences of toxicological relevance were noted in litter data and sex ratio. Statistically significant differences were noted in AGD of male foetuses of mid and high dose groups and in female foetuses of low and mid-dose groups, compared to the control. Considering that the AGD reflects the prenatal androgenic exposure, with values physiologically higher in males than in females, the increase observed in male foetuses represents a trend towards masculinisation (increase in AGD of males) and is not considered an adverse effect (i.e. decrease in AGD of males, suggesting feminisation). The same applies to the decrease observed in females, that also represents a trend towards feminisation (reduction in AGD of females) and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation). Thus, these differences were considered incidental and non-adverse effects. Furthermore, the AGD values were within the range of laboratory historical control data. At post-mortem, no treatment-related macroscopic changes were noted at low, medium and high dose. In microscopic examination, no treatment-related changes were noted in thyroid gland of females at low, medium and high dose. In external examination, small foetuses (<2.7 g) were present both in control and treated groups without a dose-relationship. One foetus in the mid-dose group had alterations classified as malformations: imperforate anus and absence of tail. These findings was considered unrelated to the test substance. In skeletal examination, malformations were detected in control and in treated groups without dose-relationship, in terms of foetuses and litters affected. The major and minor alterations detected at skeletal examination were considered incidental since they were quantitatively similar between groups or occurred in small foetuses (<2.7 g). In visceral examination, malformations were observed in one foetus of Group 3 and in one foetus of Group 4 (extremely enlarged ureter in association with kidney pelvic dilatation extreme). These findings were considered incidental since they were seen in single foetuses from different litters without a dose relationship, therefore a treatment-related effect is unlikely. The other alterations (anomalies and variations) recorded were noted both in control and treated groups, with a similar incidence. Overall, the study investigator concluded that the dosage of 75 mg/kg bw/day test substance induced slight maternal toxicity without adverse effects on pregnancy and developmental outcome. The dosages of 37.5 mg/kg bw/day and 18.75 mg/kg bw/day were well tolerated. The NOAEL for maternal toxicity could be considered at 75 mg/kg bw/day. No differences of toxicological relevance were observed between control and treated groups in the foetal development. The major and minor alterations noted at external, skeletal and visceral examinations of foetuses were considered incidental and not treatment-related since they were quantitatively similar between control and treated groups with an absence of dose-related response. Under the study conditions, the NOAEL for maternal and developmental toxicity was determined to be 75 mg/kg bw/day (Liberati, 2020).

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
No macroscopic or microscopic examination of sacrificed dams
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionised
Details on exposure:
Range-Finding Study: Three mated female rabbits per group were exposed to the test substance orally at doses of 0, 25, 50, 100, 200 or 400 mg/kg/day for days 6 through 18 of pregnancy.
Definitive Study: Thirteen or 14 mated female rabbits per group were exposed to the test substance orally at doses of 0, 2, 8 and 24 mg/kg/day for days 6 through 18 of gestation. The control group was treated with deionized water only.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Days 0- 29 of gestation
Frequency of treatment:
once a day
Duration of test:
28 d
Dose / conc.:
2 mg/kg bw/day
Remarks:
0.35 mg a.i./kg bw/day
Dose / conc.:
8 mg/kg bw/day
Remarks:
1.4 mg a.i./kg bw/day
Dose / conc.:
24 mg/kg bw/day
Remarks:
8.4 mg a.i./kg bw/day
No. of animals per sex per dose:
13 female rabbits per dose
Control animals:
yes
Maternal examinations:
Range-finding study: Body weights were determined on days 0, 6, 11, 17 and 29. Food consumption was measured daily.
Definitive study: Animals were observed daily for signs of toxicity. Body weights were taken every three days during pregnancy. Food consumption was measured daily. All surviving dams were sacrificed at study termination on gestation day 29 using sodium pentobarbital.
Ovaries and uterine content:
Range-finding study: Uterine disposition of young was recorded, and corpora lutea and resorptions sites were counted.
Definitive study: An examination of the uterus, including the number corpora lutea, implantations, and resorptions was conducted. Uteri from females that appeared non-gravid were placed in 10% ammonium sulfide solution for confirmation of pregnancy.
Fetal examinations:
Range-finding study: Animals found dead were necropsied; survivors were sacrificed on day 29 of gestation and fetuses were weighed and examined microscopically.
Definitive study: At sacrifice fetuses were weighed, and examined externally for defects. Sex determination also was conducted on each fetus. Two thirds of the fetuses were examined for skeletal and 1/3 were examined for visceral abnormalities.
Statistics:
None
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No difference of growth curve of treated groups compared to controls was observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No difference of growth curve of treated groups compared to controls was observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Range-Finding Study: Morality occurred in the dams as follows: 1/3, 1/3, 2/3, 3/3, and 3/3 for the 25, 50, 100, 200, and 400 mg/kg/day groups, respectively. A decrease in body weight was observed at 50 and 100 mg/kg/day. Apparent resorptions occurred in the two surviving females at 50 mg/kg/day but the intercurrent mortality was considered to prohibit definitive judgment on a direct effect of the test substance on maintenance of pregnancy.
Definitive Study: No effects related to treatment were observed at the doses used in this study.
Number of abortions:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The rate of resorptions was considered low and comparable in all groups.
Dead fetuses:
no effects observed
Details on maternal toxic effects:
Range-Finding Study: Apparent resorptions occurred in the two surviving females at 50 mg/kg/day but the intercurrent mortality was considered to prohibit definitive judgment on a direct effect of the test substance on maintenance of pregnancy.
Definitive Study: No effects related to treatment were observed at the doses used in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
24 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects were observed at highest test dose
Remarks on result:
other:
Remarks:
NOAEL 8.4 mg a.i./kg bw/day
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
Incidence in treated groups were comparable to controls.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Range-Finding Study: An indirect embryotoxic effect based on fetal body weight was considered to have been exhibited at 50 mg/kg/day.
Definitive Study: No effects on any parameters were attributed to treatment with the test substance.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 24 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed at highest test dose
Remarks on result:
other: NOAEL: 8.4 mg a.i./kgbw/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
24 mg/kg bw/day
Treatment related:
no
Conclusions:
Under study conditions, the NOAEL of the read across substance for maternal and embryotoxic effects/teratogenicty was determined to be 24 mg/kg bw/day (i.e., equivalent to 8.4 mg a.i./kg bw/day)
Executive summary:

A study was conducted to determine the teratogenicity of read across substance, C12-14 TMAC (35% active in dobanol 45E7), according to the method comparable to OECD 414. Based on the results of a range-finding study, thirteen or 14 mated female rabbits per group were exposed to the read across substance orally at doses of 0, 2, 8 and 24 mg/kg bw/day (i.e., 0.35, 1.4 and 8.4 mg a.i./kg bw/day) during gestation period 6 to 18. The control group was treated with deionized water only. Animals were observed daily for signs of toxicity. Body weights were taken every three days during pregnancy. Food consumption was measured daily. All surviving dams were sacrificed at study termination on gestation Day 29 using sodium pentobarbital. An examination of the uterus, including the number corpora lutea, implantations, and resorptions was conducted. Uteri from females that appeared non-gravid were placed in 10% ammonium sulfide solution for confirmation of pregnancy. At sacrifice fetuses were weighed and examined externally for defects. Sex determination also was conducted on each fetus. Two thirds of the fetuses were examined for skeletal and 1/3 were examined for visceral abnormalities. No significant maternal or fetal effects related to treatment were observed at the tested doses. Under study conditions, the NOAEL of read across substance for maternal and embryotoxic effects/teratogenicty was determined to be 24 mg/kg bw/day (i.e., equivalent to 8.4 mg a.i./kg bw/day) (TRS (HPV), 2001). Based on the results of the read across study, similar absence of development effects can be expected for the test substance.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
short exposure time i.e., on gestation Days 7-18 only
Principles of method if other than guideline:
Pre-natal development toxicity of the test substance was evaluated in rabbits following 2-h daily topical exposure at concentrations of 0.5-2% during GD 7-18.

GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE:
Shaved dorsal area.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsed with water and dried.
- Time after start of exposure: 2 h.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0, 0.5, 1.0 and 2.0%
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Days 7 - 18 of gestation.
Frequency of treatment:
Once daily (2 hours).
Duration of test:
Days 0 - 29 of gestation.
Remarks:
Concentrations: 0, 0.5, 1.0, or 2.0% (i.e., equivalent to 0, 10, 20 and 40 mg/kg bw/day, respectively)
No. of animals per sex per dose:
20 pregnant females per dose.
Control animals:
yes, concurrent vehicle
Maternal examinations:
Dams were observed twice daily for signs of toxicity, including skin irritation from Days 7 through 29. Body weights were taken on gestation Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. Individual food consumption was measured daily. A gross necropsy was conducted on dams that died in an attempt to determine the cause of death. All surviving dams were sacrificed at study termination on gestation Day 29. An examination of the uterus (including the number and location of live and dead foetuses, early and late resorptions, and implantation sites) and ovaries (including the number of corpora lutea) was conducted. Following removal of the foetuses the abdominal and thoracic cavities and organs of the dams were examined. Uteri from females that appeared non-gravid were placed in 10% ammonium sulphide solution for confirmation of pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
Body weight changes and food consumption and number of early and late resorptions, dead foetuses, total implantations, corpora lutea, skeletal abnormalities, and mean fetal body weight were compared by analysis of variance (Bartlette's). If variance was not significant, then treatment-control comparisons were made using the least significant difference (LSD) criterion. If variance was significant, then comparison was made using the t-test for unequal variances and the Wilcoxon, Mann-Whitney rank sum test. Additionally, a regression and lack of fit were performed on each of these parameters. The number of pregnancies per group, the percentage of skeletal abnormalities and soft tissue malformations were analysed by Fisher's exact test.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
Skin irritation was observed at all doses with the severity and duration of erythema, oedema, desquamation, atonia and coriaceousness increased in a dose-dependent manner.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two control, one intermediate and one high dose doe died during the study. The cause of death could not be determined. Two of the does that died aborted prior to death (one control and one intermediate dose group animal).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in congested lungs was observed for the high dose group at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Two additional abortions occurred, one each in the intermediate and high dose groups. None of these deaths or abortions were considered related to test substance toxicity.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Maternal toxic effects: no test substance related significant effects.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: no significant treatment-related maternal toxic effects were observed up to the highest tested dose.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: no significant treatment-related foetal development effects were observed up to the highest tested dose.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects
Details on embryotoxic / teratogenic effects: The incidence of foetal malformations, as well as genetic and developmental variations in the treated groups were comparable to that of the control group.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects observed up to the highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no developmental effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for maternal as well as development toxicity in rabbits were determined to be at 40 mg/kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity / teratogenicity of the test substance, C16 TMAC (25% active in water), according to a method similar to OECD Guideline 414, in compliance with GLP. This experiment was performed in New Zealand White rabbits. Twenty mated female rabbits per group were exposed topically (daily for 2 h) from Days 7 to 18 of gestation at concentrations of 0, 0.5, 1.0, or 2.0% (equivalent to 0, 10, 20 and 40 mg a.i./kg bw/day, respectively). The control group was treated with deionised water only. Clinical condition and reactions to treatment were recorded at least once daily. Body weights were recorded on Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation. All surviving females were sacrificed on Day 29 of gestation and the foetuses were removed by caesarean section. At necropsy the females were examined macroscopically. Live foetuses were weighed, sexed and were examined for visceral and skeletal abnormalities. Two control animals, one intermediate and one high dose died during the study. Two of the rabbits that died, aborted prior to death (one control and one intermediate dose). Two additional abortions occurred, one each in the intermediate and high dose groups. Deaths or abortions were not considered to be related to the test substance. No treatment-related maternal body weight or food intake effects were noted. The incidence of foetal malformations, as well as genetic and developmental variations in the treated groups was comparable to that of the control group. No other treatment-related effects were noted. Under the study conditions, the NOAEL of the test substance for maternal as well as developmental toxicity was established at 40 mg a.i./kg bw/day in rabbits (TRS (HPV), 2001).

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Strain: CFY
Details on test animals or test system and environmental conditions:
- Four groups (caged individually)
- Temperature: 20°C +/- 2°C, Relative humidity: 55 +/- 10%
- 12/12 hours dark/light
-Free access to food (Spratts Laboratory Animals Diet No 1) and tp water
Route of administration:
dermal
Vehicle:
other: distilled water
Details on exposure:
On Days 4 or 5 of pregnancy, an area (4x4 cm) of fur in the scapular region was removed by electric clippers. (Shaving was repeated when necessary but no depilatory agents were used). Dosing started on Day 6 and continued up to and including Day 15 of gestation. On each day, the test substance was applied to the shaved area at the rate of 0.5 mL/rat, and at a speed minimising 'run off' into surrounding fur. The exposed area was neither washed nor occluded at any time.
Analytical verification of doses or concentrations:
yes
Remarks:
no further details
Duration of treatment / exposure:
From Gestation Day 6 to 15
Frequency of treatment:
each day
Dose / conc.:
0 other: %
Remarks:
corresponding to 0 mg/kg bw/day
Dose / conc.:
0.9 other: %
Remarks:
corresponding to 18 mg/kg bw/day
Dose / conc.:
1.5 other: %
Remarks:
corresponding to 30 mg/kg bw/day
Dose / conc.:
2.5 other: %
Remarks:
corresponding to 50 mg/kg bw/day
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Concentrations: 0.0, 0.9, 1.5, and 2.5% in distilled water administered per 0.5 mL/rat/day (2 mL/kg bw/day for a weight of ca. 250g).
Maternal examinations:
Signs of systemic reaction and local reaction of the exposed area of skin (scores: 1-4): daily.
Body weights: on Days 1, 3, 6, 10, 17, and 20 of pregnancy.
Food and water consumption: at regular intervals throughout the study.
Ovaries and uterine content:
On Day 20 of pregnancy:
- congenital abnormalities and macroscopic pathological changes in maternal organs
- ovaries and uteri: number of corpora lutea, number/distribution of live young and embryo/foetal deaths (early and late, stages of implantation) and post implantation loss ((No. of implantations - No. of live young) x 100 / No. of implantations)
Fetal examinations:
On Day 20 of pregnancy:
- litter weight from which the mean pup weight was calculated, foetal abnormalities (external and internal + classification) and post implantation loss ((No. of implantations - No. of live young) x 100 / No. of implantations)
- sex determination
Statistics:
- Group mean values were calculated from individual litter values and, where expressed as a percentage, were calculated as the mean of the percentages within individual litters (and not from group totals for foetuses)
- Non-parametric methods (analysis of litter and abnormalities), analysis of variance (body weight, food or water consumption), and Kruskal-Wallis test (where significant heterogenicity was recorded)
Clinical signs:
no effects observed
Description (incidence and severity):
No systemic signs of reaction
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
- In terms of incidence of animals affected, and degree of reaction.
- In all cases, initial reaction was evident within a day of the first administration. It reached a peak of severity around the mid point of the dosing period, and stabilized or showed a decline thereafter. This phenomenon was frequently associated with scab formation.
- Local reactions (erythema and oedema).
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
no macroscopic pathological changes in internal organs
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No differences in the number of corpora lutea were recorded.
Details on maternal toxic effects:
Only dermal irritation was observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effect
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: overall no systemic toxicity except "dermal irritation"
Key result
Dose descriptor:
NOAEL
Remarks:
local effect
Effect level:
18 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
dermal irritation
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: dermal
Description (incidence and severity):
- In terms of incidence of animals affected, and degree of reaction.
- In all cases, initial reaction was evident within a day of the first administration. It reached a peak of severity around the mid point of the dosing period, and stabilized or showed a decline thereafter. This phenomenon was frequently associated with scab formation.
- Local reactions (erythema and oedema).
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean foetal weight was not modified by the treatment
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The litter weight was not modified by the treatment
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
In all groups, the incidence of affected litters and foetuses was within the limits of historical control values. The types of malformations or anomaly observed were compatible with the types of abnormality recorded among concurrent or historical control vlaues and there was no unusual clustering of a particular type of abnormality in any one test group.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Under the study conditions, a dermal application of the read across substance did not induce toxic effects on dams or foetuses, except for a local irritation (erythema and oedema) on the skin of pregnant animals. The maternal NOAELs (systemic and local) were therefore established at 50 and 18 mg/kg bw/day, respectively. The developmental NOAEL was determined at 50 mg/kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity of the test substance, C18 TMAC (98.8% purity), according to a method similar to OECD Guideline 414. Pregnants rats were exposed dermally to the test substance from Day 6 to Day 15 of gestation. Volumes of 0.5 mL were applied on shaved skin. The concentrations of the test substance ranged from 0.0 to 2.5% (i.e., corresponding to 0, 18, 30 and 50 mg/kg bw/day). Signs of systemic reaction and local reaction of the exposed area of skin were assessed on a daily basis. Body weights were recorded on Days 1, 3, 6, 10, 17, and 20 of pregnancy. Food and water consumption were measured at regular intervals throughout the study. On Day 20 of pregnancy, congenital abnormalities and macroscopic pathological changes in maternal organs as well as in ovaries and uteri (number of corpora lutea, number/distribution of live young and embryo/foetal deaths (early and late, stages of implantation)) and post implantation loss were evaluated. Litter weights (from which the mean pup weight was calculated), foetal abnormalities (external and internal), and sex determination were also recorded on Gestation Day 20. Under the study conditions, a dermal application of the test substance did not induce toxic effects on dams or foetuses, except for a local irritation (erythema and oedema) on the skin of pregnant animals. The maternal NOAELs (systemic and local) were therefore established at 50 and 18 mg/kg bw/day, respectively. The developmental NOAEL was determined at 50 mg/kg bw/day (Palmer, 1983). Based on the results of the read across study, similar absence of development effects can be expected for the test substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Guideline compliant or equivalent studies with test and/or read across substances
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rabbit
Quality of whole database:
Good quality studies with test and/or read across substances
Additional information

Oral:  

Study 1:  A study was conducted to determine the developmental toxicity potential of C16 TMAC (purity: 98.5%), using prenatal developmental toxicity oral gavage study in female Sprague Dawley rats, according to the OECD Guideline 414, in compliance with GLP. All animals were administered the test substance during the gestation period, starting from Day 3 through Day 19 post coitum at the doses of 0, 18.75, 37.5, 75 mg/kg bw/day (25 animals in each group). Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post-mortem examination. Blood collection for haematology and hormone determination, in association with determination of the thyroid weight from all females was performed on Day 20 post coitum. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animals died during the study. A total of three females were found not pregnant at necropsy: one in the control group, one in the low dose group (18.75 mg/kg bw/day) and one in the mid-dose group (37.5 mg/kg bw/day). One low dose female had unilateral implantation in the left horn and was not pregnant in the right one. The number of females with live foetuses on Day 20 post-coitum was: 24 in the control, low and mid-dose groups and 25 in the high dose group (75 mg/kg bw/day). Piloerection was the treatment-related clinical sign observed in all treated groups with a dose-related incidence. In Group 4 this sign was accompanied by hunched posture in few females. Minor signs, such as hair-loss and damaged ear were sporadically recorded during the study and were considered incidental. The slight statistically significant decreases in body weight observed in females of Group 4 was not considered of toxicological relevance due to the limited magnitude of the change. These changes were considered related to treatment, but not adverse. Statistically significant reduction in food consumption was observed in females of Group 4 starting from Day 6 post coitum through Day 15 post coitum. No differences were recorded on Day 18 post coitum and again a slight decrease was recorded on Day 20 post coitum. This change was considered not relevant because there was a recovery between the start and the end of treatment. The polychromasia observed in all treated groups was considered to be not adverse, considering that (a) no haematological findings were observed (e.g. anaemia) (b) no changes were recorded during the in-vivo phase or at post-mortem examination (c) no treatment-related effects were observed in existing repeated dose studies (see section 5.6 of the CSR) or other development toxicity studies (see below summaries). However, a relation with the test substance could not be excluded. Monocytosis observed in females dosed at 75 mg/kg/day was considered to be incidental. In thyroid hormone determination, no differences between control and treated females were recorded. There was no effect on the thyroid weight at any dose levels, compared to the control group. Statistically significant reductions in terminal body weight, gravid uterus weight and absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), seen in females of Group 4, compared to controls were considered not adverse due to the limited magnitude of the change. No differences of toxicological relevance were noted in litter data and sex ratio. Statistically significant differences were noted in male foetuses of mid and high-dose groups (approximately + 0.8% and + 3%, respectively) and in female foetuses of low and mid-dose groups (approximately -4% and -2.7%, respectively), compared to the control. Considering that the anogenital distance (AGD) reflects the prenatal androgenic exposure, with values physiologically higher in males than in females, the increase observed in male foetuses represents a trend towards masculinisation (increase in AGD of males) and is not considered an adverse effect (i.e. decrease in AGD of males, suggesting feminisation). The same applies to the decrease observed in females, that also represents a trend towards feminisation (reduction in AGD of females) and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation). Thus, these differences were considered incidental and non-adverse effects. Furthermore, the AGD values are within the range of the CRO’s historical control data (1.70 - 3.74mm/g1/3 for males and 0.96 - 3.00mm/g1/3 for females).

At post-mortem, no treatment-related macroscopic changes were noted at low, medium and high dose. In microscopic examination, no treatment-related changes were noted in thyroid gland of females at low, medium and high dose. In external examination, small foetuses (<2.7 g) were present both in control and treated groups without a dose-relationship. One foetus in the mid-dose group had alterations classified as malformations: imperforate anus and absence of tail. These findings was considered unrelated to the test substance. In skeletal examination, malformations were detected in control and in treated groups without dose-relationship, in terms of foetuses and litters affected. The major and minor alterations detected at skeletal examination were considered incidental since they were quantitatively similar between groups or occurred in small foetuses (<2.7 g). In visceral examination, malformations were observed in one foetus of Group 3 and in one foetus of Group 4 (extremely enlarged ureter in association with kidney pelvic dilatation extreme). These findings were considered incidental since they were seen in single foetuses from different litters without a dose relationship, therefore a treatment-related effect is unlikely. The other alterations (anomalies and variations) recorded were noted both in control and treated groups, with a similar incidence.

Overall, the study investigator concluded that the dosage of 75 mg/kg bw/day test substance induced slight maternal toxicity without adverse effects on pregnancy and developmental outcome. The dosages of 37.5 mg/kg bw/day and 18.75 mg/kg bw/day were well tolerated. The NOAEL for maternal toxicity could be considered at 75 mg/kg bw/day. No differences of toxicological relevance were observed between control and treated groups in the foetal development. The major and minor alterations noted at external, skeletal and visceral examinations of foetuses were considered incidental and not treatment-related since they were quantitatively similar between control and treated groups with an absence of dose-related response. Under the study conditions, the NOAEL for maternal and developmental toxicity was determined to be 75 mg/kg bw/day (Liberati, 2020).

Study 2:A study was conducted to determine the teratogenicity of read across substance, C12-14 TMAC (35% active in dobanol 45E7), according to the method comparable to OECD 414. Based on the results of a range-finding study, thirteen or 14 mated female New Zealand Whiterabbits per group were exposed to the read across substance orally at doses of 0, 2, 8 and 24 mg/kg bw/day (i.e., 0.35, 1.4 and 8.4 mg a.i./kg bw/day) during gestation period 6 to 18. The control group was treated with deionized water only. Animals were observed daily for signs of toxicity. Body weights were taken every three days during pregnancy. Food consumption was measured daily. All surviving dams were sacrificed at study termination on gestation Day 29 using sodium pentobarbital. An examination of the uterus, including the number corpora lutea, implantations, and resorptions was conducted. Uteri from females that appeared non-gravid were placed in 10% ammonium sulfide solution for confirmation of pregnancy. At sacrifice fetuses were weighed and examined externally for defects. Sex determination also was conducted on each fetus. Two thirds of the fetuses were examined for skeletal and 1/3 were examined for visceral abnormalities. No significant maternal or fetal effects related to treatment were observed at the tested doses. Under study conditions, the NOAEL of read across substance for maternal and embryotoxic effects/teratogenicity was determined to be 24 mg/kg bw/day (i.e., equivalent to 8.4 mg a.i./kg bw/day) (TRS (HPV), 2001). Based on the results of the read across study, similar absence of development effects can be expected for the test substance. 

Further, another pre-natal study with laurtrimonium chloride (C12 TMAC) was reported in Cosmetic Ingredient Review (CIR) report on trimoniums (Becker, 2012). This study was conducted to evaluate the development toxicity potential of the read across substance, C12 TMAC, in New Zealand White rabbits. In a range finding study, three pregnant rabbits per group were orally administered at dose levels of 0, 20, 50, 100, 200 and 400 mg/kg bw/day during gestation period 6-18. At 25 and 50 mg/kg bw/day, 1 in 3 rabbits died; at 100 mg/kg bw/day, 2 rabbits died; and at 400 mg/kg bw/day, all 3 rabbits died. There were embryonic effects (not defined) observed at 50 mg/kg bw/day. In the main study, 13-14 pregnant New Zealand White rabbits per group were orally administered 0, 2, 8, 24 mg/kg bw/day doses of C12 TMAC during gestation period 6-18. The dams were killed on Day 19 and necropsied. There were no adverse effects reported for the dams and no developmental or teratogenic effects observed (Becker, 2012).

The biocide assessment report available from RMS Italy on the read across substance, Coco TMAC, assessed the endpoint based on the above study with C12-14 TMAC (Fave 1980), Coco TMAC and DDAC and C12-16 ADBAC submitted by Lonza Cologne GmbH and Akzo Nobel Surface Chemistry AB (now Nouryon). It was concluded that the available studies with the read across quaternary ammonium compounds, show no specific potential for reproductive or developmental toxicity in rats or rabbits. Based on the Lonza studies, the maternal NOAELs in rats (based on Coco TMAC) and rabbits (based on DDAC) were both reported to be at 1 mg/kg bw/day and the developmental NOAELs were at 20 mg/kg bw/day and 3 mg/kg bw/day respectively. Based on the studies submitted by Akzo Nobel (now Nouryon), the maternal NOAELs were determined at 8.4 mg/kg bw/day (based on C12-14 TMAC), 4 mg/kg bw/day (based on DDAC) and 3 mg/kg bw/day (based on C12-16 ADBAC) and the developmental NOAELs were at 8.4, 12 and 30 mg/kg bw/day respectively. It was concluded that overall, the available studies show no specific potential for reproductive or developmental toxicity and the overall NOAEL (1 mg/kg bw/day for maternal toxicity in rats and rabbits) reflects lo local effects on the gut mucosa due to gavage bolus administration and is not relevant to the determination of a systemic AOEL (ECHA biocides assessment report, 2016). Nevertheless, due to the absence of specific reproductive toxicity and the fact that the observed effects in parents relate to general toxicity which are secondary to local effects, the maternal or developmental NOAELs were not considered to be relevant to the determination of a systemic DNELs.

Dermal: 

Study 1:A study was conducted to determine the developmental toxicity / teratogenicity of the read across substance, C16 TMAC (25% active in water), according to a method similar to OECD Guideline 414, in compliance with GLP. This experiment was performed inNew Zealand White rabbits. Twenty mated female rabbits per group were exposed topically (daily for 2 h) from Days 7 to 18 of gestation at concentrations of 0, 0.5, 1.0, or 2.0% (equivalent to 0, 10, 20 and 40 mg a.i./kg bw/day, respectively). The control group was treated with deionised water only. Clinical condition and reactions to treatment were recorded at least once daily. Body weights were recorded on Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation. All surviving females were sacrificed on Day 29 of gestation and the foetuses were removed by caesarean section. At necropsy the females were examined macroscopically. Live foetuses were weighed, sexed and were examined for visceral and skeletal abnormalities. Two control animals, one intermediate and one high dose died during the study. Two of the rabbits that died, aborted prior to death (one control and one intermediate dose). Two additional abortions occurred, one each in the intermediate and high dose groups. Deaths or abortions were not considered to be related to the read across substance. No treatment-related maternal body weight or food intake effects were noted. The incidence of foetal malformations, as well as genetic and developmental variations in the treated groups was comparable to that of the control group. No other treatment-related effects were noted. Under the study conditions, the NOAEL of the read across substance for maternal as well as developmental toxicity was established at 40 mg a.i./kg bw/day in rabbits (TRS (HPV), 2001). Based on the results of the read across study, similar absence of development effects can be expected for the test substance. 

Study 2: A study was conducted to determine the developmental toxicity of the read across substance, C18 TMAC (98.8% purity), according to a method similar to OECD Guideline 414. This experiment was performed inCFY rats.Twenty mated female rats per group were exposed dermally to the read across substance from Day 6 to Day 15 of gestation. Volumes of 0.5 mL were applied on shaved skin. The concentrations of the read across substance ranged from 0.0 to 2.5% (i.e., corresponding to 0, 18, 30 and 50 mg/kg bw/day). Signs of systemic reaction and local reaction of the exposed area of skin were assessed on a daily basis. Body weights were recorded on Days 1, 3, 6, 10, 17, and 20 of pregnancy. Food and water consumption were measured at regular intervals throughout the study. On Day 20 of pregnancy, congenital abnormalities and macroscopic pathological changes in maternal organs as well as in ovaries and uteri (number of corpora lutea, number/distribution of live young and embryo/foetal deaths (early and late, stages of implantation)) and post implantation loss were evaluated. Litter weights (from which the mean pup weight was calculated), foetal abnormalities (external and internal), and sex determination were also recorded on Gestation Day 20. Under the study conditions, a dermal application of the read across substance did not induce toxic effects on dams or foetuses, except for a local irritation (erythema and oedema) on the skin of pregnant animals. The maternal NOAELs (systemic and local) were therefore established at 50 and 18 mg/kg bw/day, respectively. The developmental NOAEL was determined at 50 mg/kg bw/day (Palmer, 1983). Based on the results of the read across study, similar absence of development effects can be expected for the test substance. 

Overall, based on the available oral and dermal pre-natal development toxicity studies with the test and/or read across substances in rats and rabbits, indicate no concern for development toxicity. Furthermore, in line with the biocides assessment report on Coco TMAC or C12 -16 ADBAC, the maternal NOAELs from the pre-natal studies have not been considered further for risk assessment.

Justification for classification or non-classification

Based on the results of the two-generation reproductive toxicity study with read across substance and pre-natal development toxicity studies with the test as well as read across substances in rats and/or rabbits, the test substance does not warrant a classification according to the EU CLP criteria (Regulation 1272/2008/EC).​  

Additional information