Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose toxicity with reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2012 to 01 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to relevant testing guidelines. A Klimisch score of 2 is assigned, as analytical verification of test concentrations was not possible due to low recoveries.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

analytical verification of test concentrations was not possible due to low recoveries.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were healthy male and female Crl:WI(Han) rats, obtained from Charles River (UK) Ltd., Margate, UK. The range-finding animals were approximately 10-12 weeks of ages and weighed 293.3 to 361.1 g (males) and 189.9 to 206.4 g (females) at the start of dosing. The main study animals were approximately 10-12 weeks of age and weighed 293.5 to 348.0 g (males) and 175.7 to 219.5 g (females) at the start of dosing.
The animals were housed in a single, exclusive room, air-conditioned to provide 15 to 20 air changes/hour and maintained at a temperature of 20 to 24°C and a relative humidity of 45 to 65%. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours darkness. The animals were housed in groups of three during the range-finding phase. Main study animals were housed in groups of up to four (pre-pairing and post pairing), one female with one male (pairing) and the females were housed individually once mated. In addition, relevant animals were housed individually for approximately 24 hours prior to FOB assessment.
SQC Rat and Mouse Breeder Diet No 3, Expanded, (Special Diets Services Ltd, Witham) and mains water was provided ad libitum.
All animals were given an inspection for ill health on arrival, and acclimatised for 15 days.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Formulations were prepared daily as a suspension in corn oil, and administered by gavage. Dose volumes were 5 mL/kg; individual dose volumes were based on individual body weights. Formulations were stored at room temperature in a sealed container, and were stirred continuously before and throughout dosing.

Details on mating procedure:

One male was paired with one female of the same treatment group for up to 10 days. One control female did not show signs of pairing after 10 days and was re-paired with a proven male of the same treatment group. Mating was confirmed by the presence of sperm in a vaginal smear or retained vagnal plug. The day of confirmation of mating was designated as Day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On Days 1 and 8 of dosing in the range-finding study, two aliquots were taken at random from the control/vehicle formulations and three aliquots were taken from the top and bottom of the test material formulation. Samples were stored at <-10°C pending possible analysis. On Days 1, 22 and 42 of the main study, two aliquots were taken at random from the control/vehicle formulations and three aliquots were taken from the top and bottom of the test material formulation. Samples were stored at <-10°C and dispatched to Covance Laboratories Inc., Madison, Wisconsin for analysis.

Duration of treatment / exposure:

Range-finding: 14 days
Main study: Males were dosed daily for 2 weeks prior to pairing, during the pairing period and a further 2 weeks before necropsy; a total of 6 weeks treatment prior to necropsy. Females were dosed once daily for 2 weeks prior to pairing, during the pairing period and until Day 4 post-partum inclusive (7 weeks prior to necropsy). The females were allowed to litter and rear their offspring to Day 4 post-partum. Dosing was deferred or omitted if the dam was in or near parturition.

Frequency of treatment:

Once daily.

Details on study schedule:

Animals were administered the test material for 2 weeks prior to mating. They were allowed up to 10 days for mating. All parental females were allowed to litter and rear offspring to Day 4 post-partum. The day pups were first observed was designated Day 0 post-partum. The date of parturition was recorded and the duration of gestation was calculated.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
other: nominal, range-finding study

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
other: nominal, main study

No. of animals per sex per dose:

Range-finding: 3/sex/dose
Main study: 10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were assigned to treatment groups on arrival using a total randomisation procedure.
Dose levels for the range-finding study were based on the results of the acute oral toxicity study in female rats, in which there were no deaths or clinical signs of toxicity following a single oral dose of 2000 mg/kg bw. During the range-finding study, daily administration was generally well-tolerated with no remarkable clinical signs at any dose level. Mean body weight gain and food consumption were reduced at 300 and 1000 mg/kg bw/d. There were no remarkable findings at necropsy and no adverse effects on haematology or clinical chemistry. Based on these findings, the same dose levels were considered suitable for the main phase.

Positive control:

Not required.

Parental animals: Observations and examinations:

Observations were made of mortality and clinical signs throughout the study. Body weight and food consumption were recorded and functional observation battery, urinalysis, haematology and clinical chemistry analyses were performed. These investigations are presented in Section 7.5.1.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Qualitative testis staging was performed.

Litter observations:

The following data were recorded for each litter: number of pups born (live and dead), daily live litter size and sex (reported on Days 1 and 4), daily clinical observations, individual pup weights on Day 1 and 4 post-partum. In addition, daily records of mortality and changes in litter size were maintained.

Postmortem examinations (parental animals):

Gross necropsy and histopathology were performed on adult animals, these investigations are presented in Section 7.5.1. The number of implantation sites were recorded for each female.

Postmortem examinations (offspring):

Gross necropsy was performed at study termination, and where possible, pups found dead or moribund were given a gross necropsy.

Statistics:

Gestation, mating, fertility and fecundity indices were analysed using the Cochran-Armitage test for dose-response and Fisher’s exact test for pairwise comparisons. The tests were interpreted with one-sided risk for decreased incidence with increasing dose. A significant trend (P<0.05) was only reported where none of the pairwise comparisons was significant. The number of implantation sites, number of pups born, percentage of male pups Day 1, pup weights, post implantation survival indices, live birth indices and viability 1 indices were analysed using non-parametric methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant, the pairwise comparisons were not reported in order to protect the Type I error.

Reproductive indices:

The following indices were used to evaluate reproductive function:
Mating index (no. females with determined copulations/no. oestrus cycles required for their insemination x 100);
Female fecundity index (no. pregnant females/no. females mated x 100);
Male fecundity index (no. males siring one or more pregnancies/no. males with one or more confirmed matings x 100);
Female fertility index (no. pregnant females/no. females paired x 100);
Male fertility index (no. males siring one or more pregnancies/no. males paired x 100)
Median pre-coital time (time (day) by which half the females in the group had shown evidence of mating)
% pre-implantation loss (no. corpora lutea - no. implantations/no. corpora lutea x 100);
% post-implantation loss (no. implantations - no. live embryos/no. implantations x 100);
% male foetuses (no. male foetuses/no. foetuses of determined sex x 100);
Gestation index (no. females with live pups/no. pregnant females x 100);
% post implantation survival index (no. pups born/no. implantation sites x 100);
% live birth index (no. pups alive Day 1/no. pups born x 100).

Offspring viability indices:

Viability index 1 (no. pups alive Day 4/no. pups alive Day 1 x 100).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Noisy respiration was observed in males and females given 300 or 1000 mg/kg bw/day on several occasions during the dosing period. No correlates were noted at necropsy or microscopic examination therefore it was concluded that this effect was not a direct clinical effect of the test material. Mouth rubbing, salivation and/or paddling of the forelimbs were noted in animals given TOFA_DimerFA_TETA_PAA from immediately post-dose up to the end of the of the day on occasion. The number of animals affected and the duration of the reactions were dose-related. These findings were considered to be a reaction to the adverse taste of the test material. In control animals, mouth rubbing and salivation immediately after dosing was noted in a few animals on isolated occasions during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related mortalities. One female in the 1000 mg/kg bw/d was euthanised on Day 1 post-partum due to the severity of
clinical signs (laboured and noisy respiration, semi-closed eyes and sluggish behaviour). At necropsy, moderate distention of the stomach, jejunum, ileum and duodenum was observed along with a slight redness to the mesenteric lymph nodes. As this was an isolated incident and there were no clinical observations or similar macroscopic observations at necropsy for other animals that received the test material, this finding was considered to be incidental and not related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

BODY WEIGHT AND WEIGHT GAIN
There was a statistically significant reduction in mean body weight gain in 1000 mg/kg bw/d males compared to controls, over the first week of the dosing period. Mean body weight gains remained slightly lower than the control group values throughout the dosing period, with a statistically significant reduction for the overall dosing period. There were however, no clinical signs for the males associated with poor clinical condition such as perinasal or peri anal hair staining and there was no effect on mating libido compared with the controls. Therefore lower body weight gain over the six week study period was considered not to have been adverse. There was no treatment related effect on body weight gain for males in the 100 or 300 mg/kg bw/d groups. At 100 mg/kg bw/d, although there was a statistically significant reduction in body weight gain for the dosing period compared with the control group value, due to the lack of a dose-response this was considered to be incidental and not an effect of treatment.
There were no effects of treatment on mean body weight and body weight gain in females during the pre-pairing period. At 100 and 300 mg/kg bw/d, there was no adverse effect of administration on mean maternal body weight gain during gestation or lactation. Over Days 1 to 4 of lactation for females that received 300 mg/kg bw/d there was a statistically significant reduction compared with the control group value. However due to the lack of a
dose-response, this was considered to be incidental and not an effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reductions in 1000 mg/kg bw/d males and females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

mesenteric lymph node; 1000 mg/kg bw/d males and females

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

FOOD CONSUMPTION
Males in the 1000 mg/kg bw/d group exhibited lower mean food consumption during Week 1 of the pre-pairing period (-16.8%). Thereafter mean food consumption improved and was similar to controls. After the pairing period, mean food consumption was similar to the control group values. Lower mean food consumption for males was transient being apparent in Week 1 only and therefore was considered not to be adverse. There were no effects on food consumption in 100 and 300 mg/kg bw/d males. There were no adverse effects on food consumption for treated females during the pre-pairing period. During the first week of the pre-pairing period, mean consumption in treated groups was lower than the control (-3.7%, -13.6% and -10% in groups given 100, 300 or 1000 mg/kg bw/d, respectively) with no dose-relationship. During the second week of the pre-pairing period, mean food consumption values were generally similar to the controls in the groups given 100 or 1000 mg/kg bw/d, with values at 300 mg/kg bw/d remaining lower than the control (-13.4%). Food consumption in treated groups was similar to that of the controls during gestation and lactation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on mating, fertility or fecundity indices. The majority of animals mated within one oestrus cycle. In the control group, one male and female did not mate during the 10 day initial pairing period. The female was re-paired with a proven male and mated successfully. The single non-mating pair was within the background range for mating at the test facility, and was therefore not considered to be related to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males in the 1000 mg/kg bw/d group exhibited decreased mean heart weight, however this finding was not considered to be adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no effects of treatment noted at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the mesenteric lymph node, increased histiocyte foci were present in all male and most female rats treated with 1000 mg/kg bw/d. Histiocyte foci were characterised by discrete aggregates of plump eosinophilic macrophages. There were no other treatment related microscopic findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose level

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
There was no adverse effect of treatment on the duration of gestation, the mean number of implantation sites or pups born, pup survival or pup body weight gain. In the groups given 100 or 1000 mg/kg bw/d, there was a statistical significant reduction in the number of pups born compared with the control group value. However, there was no dose-relationship and this was considered likely to be due to individual animal variation and a reflection of the small group sizes, rather than a treatment-related effect.

GROSS PATHOLOGY (OFFSPRING)
There were no effects of treatment noted at gross necropsy.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Table 1. Group mean litter data

	Treatment Group
	0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Number females with live pups at Day 4 post-partum	10	10	10	9
Mean duration gestation (days)	22.4	22.6	22.5	22.4
Mean number implantation sites	12.8	11.5	12.0	10.6
Mean number pups born	12.3	10.2*	11.4	9.2*
Mean number pups alive Day 1	12.3	9.9	11.4	9.2
Mean % male pups Day 1	50.6	50.7	44.2	47.2
Mean number of pups alive Day 4	12.0	9.9	11.2	9.2
Post-implantation survival index %	96.2	89.0	94.4	84.9
Live birth index %	100	97.5	100.0	100.0
Viability index %	97.0	100.0	98.3	100.0

* p<0.05

Conclusions:

There were no effects of treatment on reproductive function or pup viability, therefore the NOAEL is considered to be 1000 mg/kg bw/d.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in male and female Crl:WI(Han) rats, according to OECD Test Guideline 422. The test material, TOFA_DimerFA_TETA_PAA, was administered orally by gavage. A range-finding study was conducted with groups of 3 rats/sex. The test material was administered daily by gavage for 14 days at dose levels on 0, 100, 300 and 1000 mg/kg bw/d. Based on the findings, dose levels of 100, 300 and 1000 mg/kg bw/d were selected for the main study.

In the main study, groups of 10 male rats were dosed once daily for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. Males were treated for a minimum of 6 weeks prior to necropsy. Groups of 10 female rats were dosed for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive at total of approximately 7 weeks. The females were allowed to litter and rear their offspring to Day 4 post-partum.

There was no effect of test article administration on mating, fertility or fecundity indices; the majority of animals mated within one oestrus cycle. There was no adverse effect of treatment with TOFA_DimerFA_TETA_PAA on gestational length, number of implantation sites or pups born, pup survival or body weight gain. Pup necropsy data were unremarkable. The no-observed-adverse-effect-level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/d in males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable screening study providing multiple endpoints.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The effects of Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine on fertility have been investigated in a combined repeated dose/reproductive screening toxicity study conducted according to OECD Test Guideline 422 (Perks, 2013).

In the study groups of male and female Crl:WI(Han) rats (10 animals/group) were administered with the test substance orally by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day. Dose levels were selected based on the results of a 14-day range finding study. Male rats were dosed once daily for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. Males were treated for a minimum of 6 weeks prior to necropsy. Female rats were dosed for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive at total of approximately 7 weeks. The females were allowed to litter and rear their offspring to Day 4 post-partum.

There were no treatment related deaths during the study. No treatment-related clinical signs, changes in bodyweight gains, histopathological changes or functional changes were observed in the study. The No-Observed-Adverse-Effect-Level (NOAEL) for the general or systemic toxicity in male and female rats was considered to be 1000 mg/kg bw/day (i.e the highest dose tested).

 

No developmental effects, effects on reproductive parameters or treatment-related signs of systemic toxicity were observed in the study. No treatment-related effects were observed on mating, fertility or fecundity indices; the majority of animals mated within one oestrous cycle. No treatment-related adverse effects were observed on gestational length, the number of implantation sites or pups born, pup survival or body weight gain. Pup necropsy data were unremarkable. Based on this study, The No-Observed-Adverse-Effect-Level (NOAEL) for the effects of the substance on fertility in male and in female rats and for developmental toxicity was considered to be 1000 mg/kg bw/day (i.e. the highest dose tested).

There were no adverse findings from a 90-day repeated dose oral toxicity study or a pre-natal developmental toxicity study that highlight any reproductive hazard potential of Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine. The NOAEL for repeat dose toxicity was 1000 mg/kg bw/d on the OECD 408 90 -day Repeat dose toxicity study, with no effect on the reproductive organs.

In accordance with Column 2 of Annex X of the REACH regulation, a waiver is proposed for the extended one generation reproductive toxicity study. No adverse effects or indication of systemic exposure were observed in a combined repeated dose toxicity with reproduction/developmental toxicity screening test (OECD 422) at dose levels up to and including the limit dose. Also, according to Lipinski’s Rule of Five, the substance is not predicted to be bioavailable (OECD QSAR Toolbox). No additional testing is therefore considered necessary on scientific grounds and in the interests of avoiding unnecessary animal testing in accordance with EU Directive 86/609/EEC.

Short description of key information:
No evidence of an effect on the reproductive organs was seen in a combined repeated dose and reproductive/developmental screening study (OECD 422) or a 90 -day repeat dose toxicity study (OECD 408) in rats after repeated oral doses upto 1000 mg/kg bw/day.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Source of Test Material
Huntsman Advanced Materials, Ernst-Schering-Straße 14, 59192 Bergkamen, Germany

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd., Plot 4B, MN Park, Shameerpet Mandal, Turkapally Village, Medchal District, Telangana -500078, India

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For dose formulation analysis, prepared formulations were sampled at the prior to the initiation (04 Novmber 2019) and at termination (25 November 2019) of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored at room temperature in the experimental room depending upon the obtained stability results. For each set, one replicate sample was drawn from top, middle and bottom layers for each dose formulation. In case of control, one sample from middle layer was drawn and processed similar to dose formulation concentration. The dose formulation samples with back up were sent for formulation analysis to determine the concentration and homogeneity of the test item.
Samples were analyzed for Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine using analytical method validated under Study No. G18455.

Formulations were considered acceptable as the overall mean result (calculated using all the 6 replicate values) of all the layers was within
± 20.0 % of the claimed concentration and the relative standard deviation
(% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was equal to or less than 15.0 %.

The unused back up samples was discarded as the results of first set of analysis were within the acceptable limits

Details on mating procedure:

Cohabitation
During the mating period, females were cohabited randomly with males in a 1:1 ratio.
Proof of pregnancy: Vaginal smear exam / vaginal plug.
Length of cohabitation: 10 Days

Duration of treatment / exposure:

15 days

Frequency of treatment:

GD 5 to GD 19

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

24

Control animals:

yes

yes, concurrent vehicle

Details on study design:

A preliminary dose range finding study (DRF Study number N4573) in pregnant rats was carried out using 7 rats per group with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine dosed at 100, 300 and 1000 mg/kg/day along with the concurrent vehicle (corn oil) control group (Study number N4573). The rats were treated with the dose formulations by oral gavage at a dose volume of 4 mL/kg body weight from GD 5 to 19 and observed for clinical signs and mortality.

In this study, dose levels up to 1000 mg/kg/day were evaluated and were tolerated. No test item related clinical signs were noted at any dose. However, significant reduction in maternal body weight and decrease in feed consumption during treatment period was noted at the high dose of
1000 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 1000 mg/kg/day. The high dose of 1000 mg/kg/day was selected based on the decreases in group mean body weight gain and feed consumption. The mid and low-dose levels were selected in order to provide a graded response to the test item.

Based on the results of the dose range finding study and in consultation with the Sponsor, the following dose levels were selected for this definitive study.

Vehicle control (G1) - 0 mg/kg/day
Low dose (G2) - 100 mg/kg/day
Mid dose (G3) - 300 mg/kg/day
High dose (G4) - 1000 mg/kg/day

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid Uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions/early deaths
• Number of late resorptions/late deaths
• Gross evaluation of placenta

Fetal examinations:

a. Total number of fetuses
b. Number of live fetuses
c. Number of dead fetuses
d. Individual fetal body weight (g)
e. Anogenital distance (mm) from all live fetuses
f. Fetus sex -external determination based on anogenital distance and internal sex based on gonadal examination (internal sex) during visceral examination

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances are heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences are found significant.

Fetal weight for male and female fetuses was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female fetuses was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.
Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney/ Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05) were designated as * throughout the report

Historical control data:

Refer Annexure 8 of Final Report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weights and net body weight gain during the different periods of gestation (GD 0-5; GD 5-20; GD 0-20) were statistically comparable to the vehicle control group at 100 and 300 mg/kg/day.

At 1000 mg/kg/day, there was significant reduction (-23% to -29%) in net body weight gain during GD 5-20 and GD 0-20 as compared to vehicle control group.

There was significant decrease in corrected body weight at 300 mg/kg/day. However, this decrease was not considered adverse as the maternal body weights during different periods of gestation were comparable to vehicle control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of rats at 100 mg/kg/day were statistically comparable to the vehicle control group.

At 300 mg/kg/day, there was significant decrease (-9% to -13%) in food consumption during GD 11-14, GD 14-17, GD 17-20 and GD 5-20 as compared to vehicle control group.However, this reduction was not considered adverse as there was no concomitant decrease in maternal body weights.

At 1000 mg/kg/day, there was significant reduction (-8% to -15%) in food consumption during GD 5-8, GD 11-14, GD 5-20 and GD 0-20 .This reduction was considered treatment related as this was associated with reduction in maternal body weight gain during different periods of gestation.

The reduction in maternal body weight gain and food consumption at
1000 mg/kg/day was considered treatment related effect indicative of maternal toxicity

Clinical biochemistry findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological findings at necropsy in the vehicle control rats and rats treated at 100 mg/kg/day. Grossly, single incidence of stomach distended with gas was observed each at 300 (Rs9376) and 1000 (Rs9396) mg/kg/day dose level

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In this study, prenatal developmental toxicity of Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine was evaluated in Wistar rats following administration once daily by oral gavage at 0, 100, 300 and 1000 mg/kg/day during gestation days 5 to 19.

Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine up to 300 mg/kg/day had no adverse effect on maternal body weights, weight gain and food consumption. At 1000 mg/kg/day, there was significant decrease in body weight gain and food consumption.The maternal and litter parameters were comparable to vehicle control group at all the doses tested.There were no gross pathological changes at any dose level. Gross evaluation of the placenta revealed no findings. External, visceral and skeletal examination of fetuses revealed no signs of teratogenicity.

Based on the above findings, it is concluded that, Observed- Adverse- Effect- Level (NOAEL) for

• Maternal toxicity is 300 mg/kg/day, due to test item related significant reduction in mean maternal body weight and weight gain and reduction in food consumption at 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day, as fetal external, visceral and skeletal examinations revealed no signs of developmental toxicity or teratogenicity up to 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine when administered daily by oral gavage during gestation days (GD) 5 to 19 to presumed pregnant Wistar rats. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item for maternal and developmental toxicity.

The dose levels for this study were selected based on Dose Range Finding study (DRF). In DRF study, Study No. N4573, dose levels of 100, 300 and 1000 mg/kg/day were evaluated. The dose levels up to 1000 mg/kg/day were tolerated. No test item related clinical signs were noted at any dose. There was significant reduction in maternal body weight gains along with decreases in food consumption during treatment period at the highest dose of
1000 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 1000 mg/kg/day.The mid and low-dose levels of300 and 100 mg/kg/day were selected in order to provide a graded response to the test item.

A total of 96Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

 

Group Nos.	Groups	Dose volume (mL/kg)	Dose (mg/kg/day)	Concen- tration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control	4	0	0	24
G2	Low dose	4	100	25	24
G3	Mid dose	4	300	75	24
G4	High dose	4	1000	250	24

Test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine was administered at 0, 100, 300 and 1000 mg/kg/day. The control group received the vehicle (corn oil) only.A constant dose volume of 4 mL/kg body weight was administered to all groups.

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA).The stability and homogeneity of the test item in the vehicle was established under Study No. G18455at concentrations of5and500 mg/mL.The test item was found to be stable and also resuspendable in vehicle for 48 hour at 5 and 500 mg/mL concentration when stored at room temperature.

During the conduct of the experiment, homogeneity and active ingredient analysis was carried out from the dose formulation samples collectedprior toinitiationof treatmentand termination of treatment. The results of analysis of formulations were within the acceptable limits.

The following parameters and endpoints were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, fetus parameters [sex, weight, anogenital distance, and external, visceral and skeletal findings].All fetuses were examined for gross abnormalities. Approximately half the number of the fetuses from each litter were examined for visceral (soft tissue) malformations and the remaining half of fetuses were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormone levels were estimated from the blood collected at terminal sacrifice (on GD20).

 

The main findings of the study are presented below:

 

·   There were no mortalities and clinical signs at any of the doses tested. There were no gross necropsy findings at 100 mg/kg/day; while single incidence of stomach distended with gas was observed each at 300 and 1000 mg/kg/day dose level.

·   Mean body weights and gains and food consumption were significantly lower at 1000 mg/kg/day.

·   Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early resorptions, late resorptions, pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of 1000 mg/kg/day.

·   The litter parameters comprising total number of fetuses, number of live fetuses, male and female fetal weights and anogenital distance were statistically comparable to vehicle control group up to the highest tested dose of 1000 mg/kg/day.

·   Fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 1000 mg/kg/day.

·   Thyroid hormone levels (T3, T4 and TSH) , thyroid gland weights and histology of thyroid glands and gross finding (stomach distended with gas) were unaffected by treatment with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine up to the highest dose of 1000 mg/kg/day.

 

Based on the above findings, it is concluded that, Observed- Adverse- Effect- Level (NOAEL) for

 

·   Maternal toxicity is300 mg/kg/day,due to test item related significant reduction in mean maternal body weight and weight gain and reduction in food consumption at 1000 mg/kg/day.

 

·   Fetal developmental toxicity and Teratogencity is1000 mg/kg/day,as fetal external, visceral and skeletal examinations revealed no signs of developmental toxicity or teratogenicity up to 1000 mg/kg/day.

[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Source of Test Material
Huntsman Advanced Materials, Ernst-Schering-Straße 14, 59192 Bergkamen, Germany

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test Animals
Source
In-house, Department of Safety Assessment, Eurofins Advinus Limited, Post box no. 5813, Plot Nos. 21 & 22 Phase-II, Peenya, Industrial Area, Bengaluru 560 058, India

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis was carried out from samples prior to the initiation of treatment and before the termination of treatment.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored at room temperature. For each set, one replicate sample was drawn from top, middle and bottom layers for each dose formulation. In case of control, one sample from middle layer was drawn and processed similar to dose formulation concentration. The dose formulation samples along with back up samples were sent for formulation analysis to determine the concentration and homogeneity of the test item.

Samples were analyzed for Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine using analytical method validated under Study No. G18455.

Formulations were considered acceptable if the overall mean result (calculated using all the 6 replicate values) of all the layers is within ± 20.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers is equal to or less than 15.0 %.

The unused back up samples were disposed.

Details on mating procedure:

Cohabitation
During the mating period, females were cohabited randomly with males in a 1:1 ratio.
Proof of pregnancy: Confirmation of mating visually.
Length of cohabitation: 8 Days

Duration of treatment / exposure:

23 days

Frequency of treatment:

GD 6 to GD 28

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

G2 (Low Dose)

Dose / conc.:

35 mg/kg bw/day (nominal)

Remarks:

G3 (Mid Dose)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

G4 (High Dose)

No. of animals per sex per dose:

Day '0' Pregnant rabbits: 23

Control animals:

yes

yes, concurrent vehicle

Details on study design:

A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out under the study number N4574. Six rabbits per group were treated with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine. The rabbits were treated with the dose formulations by oral gavage at a dose volume of 2 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. The animals in the vehicle control group were handled in an identical manner as in the treatment groups and treated with vehicle (corn oil).

The initial doses tested in the study (DRF-I) were 100, 300, 600 and 1000 mg/kg/day, along with the vehicle control (G1). Considering treatment related deaths at all the test item treated groups, the surviving rabbits in these treatment groups were euthanized on GD 9 without further evaluation. The vehicle administration for control group was continued and these rabbits were sacrificed on GD 29. Due to treatment related deaths in DRF-I, additional rabbits were treated at 15 and 50 mg/kg/day, (DRF-II). These additional lower doses were well tolerated without any toxicity. Hence an additional higher dose of 75 mg/kg/day was tested (DRF –III) to arrive at the maximum tolerated dose.

Based on the reduction in body weight at 75 mg/kg/day and decrease in food consumption along with increase in post implantation loss at 50 and 75 mg/kg/day, the following doses are selected for the definitive study in consultation with sponsor:

Vehicle control - 0 mg/kg/day
Low dose - 15 mg/kg/day
Mid dose - 35 mg/kg/day
High dose - 75 mg/kg/day

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 29.
• Pregnancy status
• Gravid uterine weight (from all rabbits subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

Data were captured and compiled using Provantis Integrated Preclinical Software System.

The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions/deaths, pre-implantation and post-implantation loss, external, soft tissue and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon test pair wise comparisons of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

The incidence of external, visceral and skeletal findings for fetus and litter was tested using Cochran Armitage trend test and pair wise comparison was tested by Fisher’s exact test for group association.

Statistically significant differences (p<0.05) were designated as * throughout the report.

Historical control data:

Refer Annexure 6 of the final report

Clinical signs:

no effects observed

Description (incidence and severity):

• There were no mortalities at any of the doses tested.
• There was a total of three abortions observed one each in vehicle control, 15 and 75 mg/kg/day doses on GD 21 (RBa6177), GD 25 (RBa6199) and GD 21 (RBa6256) respectively

Mortality:

no mortality observed

Description (incidence):

There were no mortality at any of the doses tested.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were statistically non-significant reductions in maternal body weight gain at 75 mg/kg/day when compared to the vehicle control data.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was statistically significant reduction reductions in food consumption at 75 mg/kg/day when compared to the vehicle control data.

Clinical biochemistry findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed (including reproductive organs) in all rabbits

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

As rabbits are sensitive animals to handling and environmental stress, the observed number of abortions in the presented study were considered to have occurred incidentally and were within the historical control range.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions. Increase in late resorptions/post implantation loss can be correlated to lower food consumption and decreased body weights, indicative of maternal stress and hence considered secondary to maternal toxicity.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions. Increase in late resorptions/post implantation loss can be correlated to lower food consumption and decreased body weights, indicative of maternal stress and hence considered secondary to maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

35 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

At 75 mg/kg/day, there was significant reduction in mean fetal weights (12 to 15%) of males, females and combined sex. The reduced fetal weights were associated with decrease in uterine weight which may be due to reduced maternal body weight and food consumption caused by general toxicity rather than a direct developmental effect. The reduction in fetal weights (upto 15%) was within the historical control data range and is considered secondary to maternal toxicity. No test item related changes were observed in other litter parameters of total number of fetuses and mean litter size.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Fetal external examination revealed no signs of teratogenicity up to the highest tested dose of 75 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 75 mg/kg/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

Fetall visceral examination revealed no signs of teratogenicity up to the highest tested dose of 75 mg/kg/day

Key result

Dose descriptor:

NOAEC

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No affects in abscence of maternal toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Test item had no effect on maternal body weight, weight gain, food consumption, uterine and litter parameters at the doses of 15 and 35 mg/kg/day.

At 75 mg/kg/day, there was reduction in maternal body weight gain and food consumption. The decrease in uterine weight, increase in late resorptions and lower fetal weights correlated to lower food consumption and decreased body weights indicative of maternal stress and hence were considered secondary to maternal toxicity. Gross evaluation of the placenta revealed no findings. There were no gross pathological changes at any dose level.

External, visceral and skeletal examination of fetuses revealed no signs of teratogenicity up to the highest dose of 75 mg/kg/day.

In conclution, based on the above findings, under the test conditions and doses employed in this study, it is concluded that No-Observed-Adverse-Effect Level (NOAEL) for:

• Maternal systemic toxicity is 35 mg/kg/day due tendency towards lower body weight gains and significant reduction in food consumption at
75 mg/kg/day.

• Developmental toxicity and Teratogenicity is 75 mg/kg/day as decrease in uterine weight, increase in number of late resorptions and reduction in fetal weights were considered secondary to maternal toxicity and fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the high dose of 75 mg/kg/day

Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits. This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. This study provided a rational basis for risk assessment in humans and helped to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rabbits.

 

The dose levels for this study were selected based on a Dose Range Finding study (DRF) (Study Number N4574). The initial doses tested in the study (DRF-I) were 100, 300, 600 and 1000 mg/kg/day, along with the vehicle control (G1). Considering treatment related deaths at all the test item treated groups, the surviving rabbits in these treatment groups were euthanized on GD 9 without further evaluation. The vehicle administration for control group was continued and these rabbits were sacrificed on GD 29. Due to treatment related deaths in DRF-I, additional rabbits were treated at 15 and 50 mg/kg/day, (DRF-II). These additional lower doses were well tolerated without any toxicity. Hence an additional higher dose of 75 mg/kg/day was tested
(DRF –III) to arrive at the maximum tolerated dose. The rabbits were treated with test item formulations by oral gavage from GD 6 to GD 28 and observed for clinical signs and mortality. The animals in the vehicle control group were handled in an identical manner as the animals in the treatment groups and treated with vehicle (corn oil).

 

At 75 mg/kg/day there was reduction in body weight and lower food consumption along with increase in post implantation loss at 50 and
75 mg/kg/day. Based on the results, high dose of 75 mg/kg/day was selected for the definitive study. Mid dose of 35 mg/kg/day and low dose of
15 mg/kg/day were selectedto provide a graded response to the test item.

 

In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred.

 

The test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetraminewas suspended in corn oil and administered orally from GD6to28at the dose volume of2mL/kg body weight.Rabbits in the control group werewere handled in an identical manner as the animals in the treatment groupsandweretreatedwith vehicle.

Rabbitswere observed daily for clinical signs, morbidity, mortality and body weightchanges andfood consumption.All the surviving rabbits were sacrificed on GD 29 and subjected to gross pathological examination. Gross pathological examinations were also performed for rabbits that aborted during the course of study. The uteri from all the animals sacrificed on GD 29 were removed (by laparo-hysterectomy).The uteri were weighed and examined for the number of implantation sites, early and late resorptions and number of live fetuses. The number of corpora lutea was counted. All the fetuses were individually weighed and observed for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. 

The main findings of the study are presented below:

·           There were no mortalities at any of the doses tested.

·           There was a total of three abortions observed one each in vehicle control, 15 and 75 mg/kg/day doses on GD 21 (RBa6177), GD 25 (RBa6199) and GD 21 (RBa6256) respectively. As rabbits are sensitive animals to handling and environmental stress, the observed number of abortions in the presented study were considered to have occurred incidentally and were within the historical control range.

·           There were statistically non-significant reductions in maternal body weight gain and significant reductions in food consumption at 75 mg/kg/day when compared to the vehicle control data.

·           There were no gross pathological changes observed (including reproductive organs) in all rabbits.

·           At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions and significant reduction in mean fetal weights (12 to 15%) of males, females and combined sex. These changes were associated with decrease in maternal body weight gain and food consumption indicating general toxicity and hence considered secondary to maternal toxicity.Gross evaluation of placenta revealed no remarkable findings.

·           Fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the highest tested dose of 75 mg/kg/day. 

 

 

 

 

 

In conclusion, based on the above findings, under the test conditions and doses employed in this study, it is concluded that No Observed-Adverse-Effect Level (NOAEL) for:

 

·           Maternal systemic toxicity is 35 mg/kg/daydue to tendency towards lower body weight gains and significant reduction in food consumption at 75 mg/kg/day.

 

Developmental toxicity and Teratogenicity is 75 mg/kg/dayas decrease in uterine weight, increase in number of late resorptions and reduction in fetal weights were considered secondary to maternal toxicity and fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the high dose of 75 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Study was conducted to the relevant guideline and in a GLP compliant laboratory, with certificate

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No effects on developmental parameters were observed in a combined repeated dose and reproductive/developmental toxicity study conducted using Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine following oral doses upto 1000 mg/kg bw/day (Perks, 2013). Based on this study, the NOAEL for developmental toxicity was considered to be 1000 mg/kg bw/day (i.e. the highest dose tested)

A pre-natal developmental toxicity study using Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine in the rat (OECD Test Guideline 414) was conducted to evaluate the developmental hazard potential of the substance. No reproductive toxicity was observed in a combined repeated dose and reproductive and developmental toxicity screening study (OECD Test Guideline 422) conducted using Dimer trimer FA TETA PAA. In the study, rats were administered the substance by oral gavage at 0, 100, 300 or 1000 mg/kg bw/day. No signs of reproductive or developmental toxicity were observed at doses up to 1000 mg/kg bw/day. The NOAEL for effects on fertility and developmental effects was considered to be 1000 mg/kg bw/day (i.e. the highest dose tested).

No developmental toxicity was observed in prenatal developmental toxicity studies in the rat or rabbit (OECD Test Guideline 414) conducted using Dimer trimer FA TETA PAA.

In the study conducted in the rat, the test item was administered by oral gavage at 100, 300 or 1000 mg/kg bw/d. No signs of developmental toxicity or teratogenicity were observed at doses up to 1000 mg/kg bw/d. The NOAEL for fetal developmental toxicity and teratogenicity was established as 1000 mg/kg bw/d

In the study conducted with the rabbit, the test item was administered by oral gavage at 15, 35 and 75 mg/kg bw/d. There were no signs of developmental toxicity or teratogenicity, in the absence of maternal toxicity. The NOAEL for developmental toxicity and teratogenicity was established as 75 mg/kg bw/d.

As the most sensitive endpoint, the calculations for DNEL derivation and the risk assessment have used 75 mg/kg bw/d as their starting value. Rabbits are known to be sensitive to handling and stress during repeat dose studies, and the use of this lowest NOAEL is seen as conservative for use as the starting point for risk assessment and protective of human health and the environment.

Justification for classification or non-classification

No classification is proposed: relevant data do not show need for classification.