Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose toxicity with reproduction/developmental toxicity screening test
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
The test material, TOFA_DimerFA_TETA_PAA, was described as a yellow liquid, with batch number BB001030V1, and a purity of 100%. The test material was stored in a sealed container at room temperature (15 - 25°C).

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals 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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs related to adverse taste of test material
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reductions in 1000 mg/kg bw/d males and females
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
Description (incidence and severity):
decreased mean heart weight in 1000 mg/kg bw/d males
Gross pathological findings:
no effects observed
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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
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.

CLINICAL SIGNS
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.

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
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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
body weight and weight gain
other: Duration of gestation, mean number of implantation or pups born, pup survival or pup body weight gain
Remarks on result:
other: There were no effects of treatment on reproductive function or pup viability, therefore the NOAEL is considered to be 1000 mg/kg bw/d.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.