Amides, tall-oil fatty, N,N-di-Me

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2013 to 13 November 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable, GLP and OECD Test Guideline No 422 compliant study providing results from a proprietary screening study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:male and female Wistar Hannover RccHan:WIST rats, obtained from Harlan Laboratories Models, S.L
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 346-383g; Females: 190-225g
- Fasting period before study: not applicable
- Housing:in standard laboraotory caging, group housd prior to mating; pair housed one male and one female during mating phase and housed individually subsequently
- Diet (e.g. ad libitum): Pelleted Harlan Teklad 2014C or 2018C rat/mouse maintenance diet provided ad libitum
- Water (e.g. ad libitum): Bottled tap water available ad libitum
- Acclimation period:The rats were acclimatised for at least 5 days prior to the start of the study.

Experimental dates 25 March 2013 to 13 November 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The amount of test material and administration volume was determined for each animal based on the most recent body weight. The dose volume was 4 mL/kg bw. No correction for purity was applied to the administered dose.

Dose levels were selected based on a dose range-finding study (Study S41514). Dose levels of 0, 50, 300 and 1000 mg/kg bw/day were chosen for the main study.
Oral gavage administration daily for two weeks prior to mating; males were dosed daily for at least 48 days after mating up to and including the day before sacrifice; the females were dosed up to and including the day before sacrifice (day 4 postpartum).

Details on mating procedure:

Animals were paired one male to one female for a period of 18 or 24 hours following two weeks of dosing. When evidence of copulation in the daily vaginal smear was sperm-positive, and/or a copulation plug was observed the females were removed and housed individually. If a female did not mate within the 14 day mating period, the female was paired with a proven male from the same group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots were taken from each of the dose formulations and analysed for concentration and homogeneity using GC-FID. Analyses were completed twice during treatment in the first week of treatment and during the post-pairing period. Homogeneity analyses were completed during the first week of treatment.
The results obtained showed the concentrations in the formulations ranged from 86.0% to 117.1% (within the accepted range of 80-120%) and the CV for homogeneity did not exceed 6%.

Duration of treatment / exposure:

Males: two weeks prior to mating and at least up to and including the day before sacrifice (at least a total of 28 days).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).

Frequency of treatment:

Daily

Details on study schedule:

Day 1: First treatment
Day 1 to 15: - pre-pairing treatment (checks for morbidity, bodyweight, food consumption and clinical signs)
Day 15: mating (checks for morbidity, bodyweight, clinical signs and evidence of mating - vaginal smears)
Day 16 - Day 0 of pregnancy
Day 16 to 37 - gestation (checks for morbidity, bodyweight, food consumption and clinical signs)
Day 37: Delivery
Day 38: Lactation F0 females (checks for nursing, morbidity, bodyweight, food consumption and clinical signs)
F1 litter size , sex, bodyweight, clinical signs and a behaviour test
F0 males (checks for morbidity, bodyweight and clinical signs)

Day 41: End of treatment (F0 females: day 4 postpartum (checks for grip strength, motor activity, sensory reactivity to stimuli) and F1 necropsy; F0 males (grip strength, motor activity, sensory reactivity to stimuli)

Day 42: F0 males (necropsy and blood sampling; F0 females Day 5 postpartum (necropsy and blood sampling)

Remarks:

Doses / Concentrations:
0, 50, 300, 1000 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

10 rats/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were chosen based on the 14-day range finder.
Males and females were administered the test material for 14 days prior to mating.

Positive control:

Not required.

Parental animals: Observations and examinations:

From day 21 postcoitum, the females were examined at least twice daily for signs of parturition. Gestation length was recorded.
For the F0 generation twice daily checks were made for morbidity and mortality and once daily cage-side checks of clinical signs. A detailed behavioural assessment was completed for the test and control animals at weekly intervals. A functional performance test for grip strength and motor activity, sensory reactivity assesssments for auditory, visual and proprioceptive stimuli were evaluated. Food consumption was measured for the males at weekly intervals during the pre-pairing period and during two weeks of the post-mating phase and at weekly intervals through the study for the females.
water consumption was recorded for males during day 7-15 post-pairing and for females on days 6-14 post-coitum.
Bodyweights were recorded for at weekly intervals pre-pairing and daily thereafter.
Vaginal smears were collected from all females and examined for oestrous cycling during the mating phase. Smears were discontinued when sperm were found.
The females were checked from day 21 postcoitum for signs of parturition and gestation length was recorded. The females that gave birth were observed to determine if they nursed their young.

Oestrous cyclicity (parental animals):

Vaginal smears were collected from all females during the mating period until the presence of sperm was detected.

Sperm parameters (parental animals):

A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high dose groups was performed.

Litter observations:

The number of still births, live and dead pups and any macroscopic anomalies were recorded for each litter within 24 hours of parturition. Pup sex was recorded. Checks for morbidity/mortality were made at least twice daily. Animals were observed for clinical signs at least once daily. Body weights were recorded on day 1 and 4 postpartum. The surface righting reflex of the pups was evaluated on day 1 postpartum. Presence of milk (lactation) was determined on days 1 to 4 postpartum.

Postmortem examinations (parental animals):

Gross macroscopic examination was performed of all internal organs with emphasis on the uterus, count of corpora lutea and implantation sites. If no implantation sites were found then the uterus was placed in an aqueous solution of ammonium sulfide to highlight implantation sites.

F0 females were terminated on day 5 postpartum. The mated females that did not give birth or show any signs of pregnancy (five individuals numbers 58, 62, 65, 71 and 78) were terminated 26 days postcoitum

Postmortem examinations (offspring):

Any rat sacrified or found dead during the study was examined macroscopically where possible. Surviving pups were sacrificed on day 4 postpartum and examined macroscopically. Samples of organs and tissues with macroscopic alterations were preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.

Statistics:

Dunnett test, Fisher's Exact test and Steel testwere variously used for the assessment of parameters

Reproductive indices:

Pre-coital time, percentage mating, conception rate, fertility index, pre-implantation loss, post-implantation loss, gestation length, gestation index.

Offspring viability indices:

Post-natal loss, viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was recorded in males and females from control and at 50, 300 and 1000 mg/kg bw/day, with greater incidence at 1000 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the premating period; afterwards there was a recovery in body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the premating period; afterwards there was a recovery in body weight.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocyte hypertrophy in both sexes at 1000 mg/kg bw/day and in males at 300 mg/kg bw/day

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

spermatology assessments indicated some histopathological correlates with small testes in all treated groups but with no relationship to treatment

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Corpora lutea and implantation site counts were lower at 300 and 1000 mg/kg bw/day. No differences were recorded at 50 mg/kg bw/day.

The reproductive performance showed no notable differences in number of mated females, although slightly fewer females were pregnant in the 300 and 1000 mg/kg bw/day groups and the number of females with live pups was lower in the treated dose groups showing a dose related reduction.

There were no differences in median or mean pre-coital time. There were no treatment related differences in the percentage of mating or in the gestatiion index.

The mean number of implantation sites per litter and lower corpora lutea were recorded in the 300 and 1000 mg/kg bw/day. The percent pre- and post-implantation losses were not markedly different for control and treated groups. No effects apparent in the 50 mg/kg bw/day. No differences were apparent in sex ratio.

The duration of pregnancy was similar in all groups with a mean of 22 days.

The percentage of pups with milk present in the stomach was similar in all dose groups. One female dosed at 1000 mg/kg bw/day had agalactorrhea and was unable to nurse its pups which consequently died between days 0 and 2 of lactation.

The organ weight analysis indicated higher liver weights for males dosed at 300 or 1000 mg/kg bw/day and for females dosed at 1000 mg/kg bw/day. Effects relative to bodyweight and brain weight were significantly increased in the high ddose group. Significantly higher kidney weights were apparent in males dosed at 1000 mg/kg bw/day and increases were noted for females dosed at 300 or 1000 mg/kg bw/day in thymus and adrenal.
No organ weight changes were evident in the low dose group rats.
Isolated cases of lower testes weight, affecting one male in each dose group, were noted. No effects were evident for the female reproductive organs.


The observations of small testes and /or epididymides recorded at 50 , 300 and 1000 mg/kg bw/day in individual male rats was correlated with histopathological changes recorded as bilateral tubular degeneration in the testes or oligospermia/aspermia in the epididymides. Findings are considered to be spontaneous and not related to treatment.

Minimal hepatocellular hypertrophy was noted for males but not females dosed at 300 mg/kg bw/day and no hepatocellular hypertrophy was evident in either sex dosed at 50 mg/kg bw/day. In the high dose group, slight hepatocellular hypertrophy was apparent in the male and female livers, the enlarged hepatocytes had a ground glass appearance. For both males and females there was minimal to moderate thyroid follicular cell hypertrophy. The thyroid changes were characterised by irregularly shaped follicles lining the columnar cells and containing a small amount of pale-staining colloid. Minimal cell hypertrophy was noted in the pars distalis of the pituitary of one male. The pituitary change was characterised by an increased number of scattered enlarged pale eosinophilic cells.
A greater incidence of a decrease in lymphocytes was observed in the mesenteric lymph nodes of males and females and in male thymus. An increased incidence of hyaline droplets within the proximal tubule lining of the kidneys was apparent in the male rats.
The non-pregnant female no 71 had diffuse vacuolation of the ovaries (including interstitial, luteal and follicular cells) and vacuolation of the epithelial cells lining the oviducts. Vacuolation was also present in the enlarged adrenals.
Other females showed no notable microscopic findings.

Dose descriptor:

NOAEL

Remarks:

Toxicity

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: The NOAEL for males of 50 mg/kg bw/day is based on increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at 300 mg/kg bw/d.

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The NOAEL for females of 300 mg/kg bw/d is based on reduced weight gain, increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at the highest dose level of 1000 mg/kg bw/d.

Dose descriptor:

NOEL

Remarks:

Reproductive toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The NOAEL of 50 mg/kg bw/d is based on lower implantation sites, lower corpora lutea numbers and smaller litter size at 300 and 1000 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects were seen at the highest dose level of 1000 mg/kg bw/d

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

There were no significant differences in mean pup weight on day 1 postpartum.
There were no differences in the mean number of dead pups at the first litter check and the mean number of live pups was lower in the 300 and 1000 mg/kg bw/day. The live birth index was similar for all groups. The percentage postnatal losses were similar for treated or control groups.

No differences were recorded in litter bodyweights.

No treatment related changes were apparent during the morphological examination of the pups. Head abnormalities were recorded in pup no. 7 from one control litter including absence of mouth, mandible, eyes and nares.

The postural reflexes in the pups resulted in a lower percentage of foetuses with positive responses in the surface-righting reflex in the high dose group. There were no differences in the 50 and 300 mg/kg bw/day.

Macroscopic abnormalities observed for the offspring included head abnormalities for one control pup including malpositioned brain, absence of olfactory bulbs and no recognizable eye structures or brain parts. One pup in the low dose group had a reddish thymus. No findings were observed in the 300 mg/kg bw/day. In the 1000 mg/kg bw/day group one pup had a partially haemorrhagic liver, possibly attributable to termination procedures, and considered to be within the range of normal background findings.

Dose descriptor:

NOAEL

Remarks:

Offspring toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were apparent in the offspring at the highest dose administered

Reproductive effects observed:

not specified

Effects contributing to male parental systemic toxicity NOAEL of 50 mg/kg bw/day

		Males – DMATO dose level mg/kg bw/day
		0	50	300	1000
Serum cholesterol	Mean (mmol/L) SD	2.01 0.38	1.80 0.22	2.65* 0.44	3.16** 0.17	Samples from 5 rats at end of treatment
Bodyweight	Mean (g) SD	441.2 17.0	440.8 13.8	416.4** 19.2	399.5** 15.0	N= 10
Liver weight	Mean (g) SD	10.38 0.80	10.80 1.24	11.35 0.49	14.18** 0.90	N= 10
Liver/bodyweight ratio	Mean (%) SD	2.35 0.14	2.45 0.21	2.73** 0.13	3.55** 0.21
Kidney weight	Mean (g) SD	2.31 0.15	2.41 0.31	2.40 0.15	2.56* 0.15	N= 10
Kidney/bodyweight ratio	Mean (%) SD	0.52 0.04	0.55 0.06	0.58* 0.04	0.64** 0.05
Hepatocyte hypertrophy minimal centrilobular slight - diffuse		-- --	-- --	4 --	-- 9	N= 10
Thyroid follicular cell hypertrophy minimal slight moderate		-- -- --			-- 3 2	N= 5
Hyaline droplets in renal tubular epithelium minimal slight		1			5	N= 5

*/** Dunnett test , pooled variance significance levels at 5% or 1%

Effects contributing to female parental systemic toxicity NOAEL of 300 mg/kg bw/day

		Females – DMATO dose level mg/kg bw/day
		0	50	300	1000
Serum cholesterol	Mean (mmol/L) SD	1.88 0.43	2.56 0.42	2.43 0.47	2.89* 0.73	Samples from 5 rats at end of treatment
Bodyweight	Mean (g) SD	245.4 18.5	248.3 13.6	251.6 19.8	245.0 11.0	N= 10
Weight gain	Pre-mating (g)	23.0	17.1	17.3	18.8
	Pregnancy (g)	108.0	108.6	109.6	99.0
	Lactation (g)	18.7	23.8	14.0	7.3
Liver weight	Mean (g) SD	8.42 0.58	8.62 1.26	8.66 1.07	10.82** 1.85	N= 10
Liver/bodyweight ratio	Mean (%) SD	3.44 0.19	3.48 0.54	3.45 0.39	4.41** 0.72
Hepatocyte hypertrophy minimal centrilobular slight - diffuse		-- --	-- --	-- --	1 4	N= 10
Thyroid follicular cell hypertrophy minimal slight moderate		-- -- --			2 1 1	N= 5

*/** Dunnett test , pooled variance significance levels at 5% or 1%

Mean litter size at birth was lower at 300 and 1000 mg/kg bw/d; findings are secondary to lower numbers of corpora lutea and implantations. Pre-implantation and post-implantation losses were comparable in all groups.

Summary of effects on fertility and development

Dose level (mg/kg bw/d)	0	50	300	1000
Mated (#)	10	10	10	10
Pregnant (#)	10	9	9	8
Fertility index (%)	100	90	90	80
Gestation index (%)	100	90	88.8	80
Corpora lutea (#)	15.9	15.0	13.8	13.5
Implantations (#)	13.9	14.0	11.3	10.9
Live pups (Day 0)	12.7	12.6	11.4	10.3
Live pups (Day 4)	10.9	11.6	11.1	8.6
Pup weight (Day 0)	5.5	5.6	5.7	5.7
Pup weight (Day 4)	8.4	8.5	8.4	8.1

Conclusions:

A NOAEL for reproductive toxicity of 50 mg/kg bw/d can be determined for this study, based on reduced corpora lutea, implantations and litter size at dose levels of 300 and 1000 mg/kg bw/d. A NOAEL for developmental toxicity of 1000 mg/kg bw/d can be determined in the absence of any effects at the highest dose level.

Executive summary:

 This study was performed to OECD Guideline 422 [Combined repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test] with the Submission Substance DMATO. Groups of RccHan:WIST rats were gavaged with DMATO (in corn oil) at dose levels of 0 (vehicle control), 50, 300 or 1000 mg/kg bw/d at a constant dose volume of 4 mL/kg bw/d. Animals were dosed for a two week pre-mating period, throughout mating, and during pregnancy and early lactation for females). The dosing period for males was at least 49 days.

Clinical signs, behavioural assessments, body weights and food and water consumption were monitored during the study. Females were also frequently examined on days 21 and 22 of pregnancy in order to check delivery. Haematology and clinical biochemistry were evaluated at termination on five selected males and females from each dose group. Pairing of animals within each dose group was undertaken on a 1:1 basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring up to Day 4 of lactation. During the lactation phase, clinical observations were recorded daily for all surviving offspring, together with litter size and offspring weights; and surface righting reflex was assessed. Extensive functional observations were performed on five selected males from each dose group the day before sacrifice and for five selected parental females from each dose group on Day 4 post partum. Adult males were terminated at least on Day 49, adult females on Day 5 post partum and offspring on Day 4 post partum. All animals were subjected to a gross necropsy examination and selected tissues from five males and five females from groups 1 and 4 were evaluated histopathologically.

No mortality was recorded during the study period. Salivation was recorded in males and females in all groups, with a greater incidence at 1000 mg/kg bw/day. Lower locomotor activity was recorded in both sexes at 300 and 1000 mg/kg bw/day. No relevant differences from the control group were recorded in the grip strength test in either sex. Sensory reactivity assessment did not reveal any differences from the control group; all animals showed a normal behaviour and appearance.

Higher water consumption was recorded in both sexes at 1000 mg/kg bw/day; no differences were recorded at 50 and 300 mg/kg bw/day. No relevant differences from the control group were recorded in food consumption. Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the pre-mating period; afterwards there was a recovery in bodyweight. No test-item related differences from the control group were recorded at 50 mg/kg bw/day.

Haematology revealed an increase in reticulocyte values and white blood cells males at 1000 mg/kg bw/day; no relevant differences were observed in the remaining groups or females. Clinical chemistry assessment showed an increase in cholesterol and triglyceride levels at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Increases in alkaline phosphatase and alanine aminotransferase were recorded at 1000 mg/kg bw/day in males and females.

Gross necropsy revealed a reddish discoloration of the mesenteric lymph node in both sexes at 1000 mg/kg bw/day. All other gross lesions recorded were considered to be within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.

An increase in liver weight was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. Moreover, higher kidney weight was recorded in males at 1000 mg/kg bw/day. One male from each test-item-treated group had lower testis weight. No noticeable differences were observed in the remaining animals.

 

At 1000 mg/kg bw/day, cell hypertrophy in the liver, thyroid and pituitary consistent with liver enzyme induction, as well as, decreased lymphocytes in the mesenteric lymph node and thymus were recorded in both sexes. Increased hyaline droplets in the kidneys were observed in males. The hyaline droplets observed in high dose males are consistent with species specific male kidney nephropathy arising from administration of high oil content doses that may exacerbate the accumulation of alpha2μ-globulin, a unique protein occurring spontaneously in proximal convoluted tubular epithelial cells only in the mature male rat. Thus, the male rat hydrocarbon nephropathy should not be predictive of a normal human renal response. One female at 1000 mg/kg bw/day had vacuolar changes (involving ovary, oviduct and adrenals). At 300 mg/kg bw/day, cell hypertrophy in the liver was recorded in males, but not in females.

Sperm Stage Evaluation showed severe diffuse bilateral tubular degeneration, associated with marked oligospermia or aspermia in the epididymides in males that showed small testes and/or epididymides. No remarkable findings were recorded in the remaining males from high and control groups.

Smaller litter size, secondary to reduced numbers of corpora lutea and implantations, was noted at 300 and 1000 mg/kg bw/d.

For general toxicity, a NOAEL for males of 50 mg/kg bw/day is determined, based on increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at 300 mg/kg bw/d. ANOAEL for females of 300 mg/kg bw/d is based on reduced weight gain, increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at the highest dose level of 1000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable, modern guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
An OECD 422 screening study is available

Justification for selection of Effect on fertility via oral route:
Only study available for this endpoint

Effects on developmental toxicity

Description of key information

An OECD 422 screening study is available for the submission substance.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: 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, guideline and GLP compliant study

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

An OECD Guideline 422 [Combined repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test] was performed with the Submission Substance DMATO. Groups of RccHan:WIST rats were gavaged with DMATO (in corn oil) at dose levels of 0 (vehicle control), 50, 300 or 1000 mg/kg bw/d at a constant dose volume of 4 mL/kg bw/d. Animals were dosed for a two week pre-mating period, throughout mating, and during pregnancy and early lactation for females). The dosing period for males was at least 49 days.

Clinical signs, behavioural assessments, body weights and food and water consumption were monitored during the study. Females were also frequently examined on days 21 and 22 of pregnancy in order to check delivery. Haematology and clinical biochemistry were evaluated at termination on five selected males and females from each dose group. Pairing of animals within each dose group was undertaken on a 1:1 basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring up to Day 4 of lactation. During the lactation phase, clinical observations were recorded daily for all surviving offspring, together with litter size and offspring weights; and surface righting reflex was assessed. Extensive functional observations were performed on five selected males from each dose group the day before sacrifice and for five selected parental females from each dose group on Day 4 post partum. Adult males were terminated at least on Day 49, adult females on Day 5 post partum and offspring on Day 4 post partum. All animals were subjected to a gross necropsy examination and selected tissues from five males and five females from groups 1 and 4 were evaluated histopathologically.

No mortality was recorded during the study period. Salivation was recorded in males and females in all groups, with a greater incidence at 1000 mg/kg bw/day. Lower locomotor activity was recorded in both sexes at 300 and 1000 mg/kg bw/day. No relevant differences from the control group were recorded in the grip strength test in either sex. Sensory reactivity assessment did not reveal any differences from the control group; all animals showed a normal behaviour and appearance.

Higher water consumption was recorded in both sexes at 1000 mg/kg bw/day; no differences were recorded at 50 and 300 mg/kg bw/day. No relevant differences from the control group were recorded in food consumption. Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the pre-mating period; afterwards there was a recovery in bodyweight. No test-item related differences from the control group were recorded at 50 mg/kg bw/day.

Haematology revealed an increase in reticulocyte values and white blood cells males at 1000 mg/kg bw/day; no relevant differences were observed in the remaining groups or females. Clinical chemistry assessment showed an increase in cholesterol and triglyceride levels at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Increases in alkaline phosphatase and alanine aminotransferase were recorded at 1000 mg/kg bw/day in males and females.

Gross necropsy revealed a reddish discoloration of the mesenteric lymph node in both sexes at 1000 mg/kg bw/day. All other gross lesions recorded were considered to be within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.

An increase in liver weight was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. Moreover, higher kidney weight was recorded in males at 1000 mg/kg bw/day. One male from each test-item-treated group had lower testis weight. No noticeable differences were observed in the remaining animals.

 

At 1000 mg/kg bw/day, cell hypertrophy in the liver, thyroid and pituitary consistent with liver enzyme induction, as well as, decreased lymphocytes in the mesenteric lymph node and thymus were recorded in both sexes. Increased hyaline droplets in the kidneys were observed in males. The hyaline droplets observed in high dose males are consistent with species specific male kidney nephropathy arising from administration of high oil content doses that may exacerbate the accumulation of alpha2μ-globulin, a unique protein occurring spontaneously in proximal convoluted tubular epithelial cells only in the mature male rat. Thus, the male rat hydrocarbon nephropathy should not be predictive of a normal human renal response. One female at 1000 mg/kg bw/day had vacuolar changes (involving ovary, oviduct and adrenals). At 300 mg/kg bw/day, cell hypertrophy in the liver was recorded in males, but not in females.

Sperm Stage Evaluation showed severe diffuse bilateral tubular degeneration, associated with marked oligospermia or aspermia in the epididymides in males that showed small testes and/or epididymides. No remarkable findings were recorded in the remaining males from high and control groups.

Smaller litter size, secondary to reduced numbers of corpora lutea and implantations, was noted at 300 and 1000 mg/kg bw/d.

For general toxicity, a NOAEL for males of 50 mg/kg bw/day is determined, based on increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at 300 mg/kg bw/d. ANOAEL for females of 300 mg/kg bw/d is based on reduced weight gain, increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at the highest dose level of 1000 mg/kg bw/d.

Justification for selection of Effect on developmental toxicity: via oral route:
No effects were observed at 1000 mg/kg bw/day for rat offspring in a reproductive / developmental toxicity study conducted in accordance with OECD Guideline 422

Justification for classification or non-classification

The results of an OECD 422 reproductive/developmental toxicity screening study do not indicate any effect on fertility/ reproductive performance for males treated at the highest dose level, 1000 mg/kg bw/day. Female reproductive performance was affected at the high dose with observation of lower implantation sites and corpora lutea but no effects were evident on any of the developmental parameters - offspring not adversely affected by exposure to 1000 mg/kg bw/day through gestation. Observation of male rat specific effects on hyaline droplet observation in renal tubular eiptheilium was not considered relevant to human risk assessments and the observation of hepatocellular hypertrophy in high dose males and females may be indicative of an adaptation to increased liver metabolism rather than a systemic toxicity response. Conservatively the NOAEL for systemic toxicity was determined to be 300 mg/kg bw/day and as consequence classification for reproductive or developmental toxicity is not warranted.