A mixture of: isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-dodecylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-tetracosylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-5,6-didodecyl-phenol. n=5 or 6

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a one-generation-study (OECD 415) 0, 150, 400, and 1000 mg/kg/day of the test article was administered orally by gavage prior to mating and throughout mating, gestation and lactation to male and female rats. The substance did not affect reproductive processes, survival, weight or size of offspring and had no effects on food consumption or body weight gain. Also pathology was without finding. The NOEL for P and F1 generation is considered to be 1000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-02 - 2008-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

other: Crl: CD®(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent on the 28 May 2008 and the 22 July 2008, respectively to provide 96 animals of each sex for the study
- Age at study initiation: 4 weeks
- Weight at study initiation: (P) Males:81-99g; Females: 150-173g;
- Housing: The animals were initially housed two per cage. The cages were made of polypropylene, with solid bottoms and stainless steel mesh tops, and measured ca 48 x 37.5 x 21 cm. A stainless steel food hopper and polypropylene or a polycarbonate water bottle was provided for each cage. The cages were suspended on a series of racks. Male and female cages were racked separately.
A few days prior to mating, males were transferred into individual grid-bottomed cages (ca 59 x 38.5 x 20 cm) of a similar design. Excreta were collected on a tray lined with absorbent paper suspended beneath the cage. After mating, the males were housed 2 per cage with their original cage-mates in solid bottomed cages. Mated females were transferred into individual ca 48 x 37.5 x 21 cm solid-bottomed cages. Just prior to the anticipated parturition, some white paper tissue was provided as nesting material.
On Day 101 of treatment (Day 0 = First day of treatment); Animals 63 and 64 (Group 3M) were suspected of fighting and were removed from each other and housed singly; these animals were re-housed together on Day 107 of treatment.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, supplied by Special Diets Services Ltd., Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch used during the study has been retained in the study archive.
- Water (e.g. ad libitum): The animals had access to domestic quality mains water ad libitum. The supply is analyzed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate of a typical recent analysis has been retained in the study archive.
- Acclimation period: The animals were acclimatized in the Charles River animal room for a minimum of 12 days from arrival until commencement of treatment. All the animals were clinically examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 hours.

IN-LIFE DATES:
Experimental Start Date: 02 June 2008
Experimental Completion Date: 13 October 2008

- Environmental Enrichment
To provide environmental enrichment, wooden chewsticks were made available to the animals, as appropriate. The chewsticks were not considered to contain any additional substances in sufficient concentrations to influence the outcome of the study.

- Animal Identification
Each animal received a subcutaneously implanted electronic identification chip identifying it individually within the study and which corresponded to that animal’s number. It was also given a cage card which was color coded for treatment group and marked with the study number, animal number, cage number and treatment group.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at approximately weekly intervals and used within the established stability period of 8 days (established in Charles River Laboratories Study No. 424799, Method 2479). An appropriate amount of test item was weighed and placed in a suitably sized glass container, and then the appropriate amount of vehicle was added and mixed by magnetic stirring until a visibly clear, homogenous suspension was obtained. The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80. Full details of the vehicle used are retained in the study raw data. To correct for purity, a correction factor of 1.04 was used for the calculation of doses on this study.

The animals were dosed once daily by oral gavage, at a dose volume of 10 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined by the weight of the animal as measured at the time of administration, except during the fetal period of gestation (from day of 16 gestation until parturition was complete); throughout this period the dose volume was determined by the weight of the animal on Day 16 of gestation. The animals were dosed once daily, at approximately the same time each day. The F0 males were dosed for 10 weeks prior to mating. The F0 females were dosed for two weeks prior to mating. Dosing for both the sexes continued throughout mating and throughout gestation and lactation for the females.

Details on mating procedure:

- M/F ratio per cage: Animals were paired in numerical order on a 1 male to 1 female basis
- Length of cohabitation: Each female was transferred to the cage of its appropriate co-group male near the end of the working day, where it remained until mating had occurred. Each female was left with its designated male for a maximum of 14 nights.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- The time taken to show a positive mating sign and the number of failed opportunities to mate (estrous passed without sign of mating) was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations was undertaken with regard to concentration and homogeneity from formulations prepared for use on Day 1 dosing of males, Day 1 dosing of females and from Week 11 of dosing. On each occasion, ca 1mL triplicate samples were taken (top, middle and bottom) from the formulations for groups 1-3 and ca 0.5mL triplicate samples from the group 4 formulations. These were analyzed to provide data on concentration and homogeneity. The samples were assayed using methodology established under Charles River Study No. 424799, Method No.2479.
The results of the analysis of the dosing formulations were within ±9.5% of the nominal concentration indicating acceptable accuracy of formulation. The low coefficients of variation (<4%) indicated that the formulations were homogenous.

Duration of treatment / exposure:

Test item was administered for 10 weeks prior to mating for males and two weeks prior to mating for the females, then throughout mating, gestation and lactation until termination after the litter had reached Day 21 of lactation.

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were agreed with the Sponsor after evaluation of existing relevant toxicological data, including a developmental toxicity study in rats (Charles River Study No. 494929).
- Rationale for animal assignment:
Animals were allocated to treatment groups by the use of randomly sequenced numbers, in such a way that each full rack contained similar numbers of representatives from all groups.
- Other:
The spare animals were numbered 193-194 (males) and 195-196 (females). Animal 116 was killed prematurely on Day 4 of dosing. The signs for this animal were consistent with damage caused by the dosing procedure and therefore it was considered appropriate to replace this animal with Animal 195 on Day 4 of treatment of the females.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
All the animals were examined during each day, from commencement of dosing. The onset, intensity and duration of any clinical signs were recorded. Once each week (starting during the pre-trial period) all adult animals were given a detailed clinical examination, including appearance, movement and behavior patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
For both sexes, body weights of F0 animals were recorded one week prior to the first day of dosing, then daily throughout the dosing period until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Males: The weight of the food consumed was recorded weekly from one week before commencement of treatment until placement of males in individual cages prior to mating. Consumption over complete weeks was also recorded after the 14 day mating period.
- Females: The weight of the food consumed was recorded weekly from one week before the commencement of treatment until the placement of males in individual cages prior to mating. Consumption was also recorded over the periods Days 0-7, 7-14 and 14-20 of gestation, and Days 0-7 and 7-14 of lactation.

OTHER:
Any deficiencies in maternal care were recorded. Points looked for included inadequate construction and cleaning of the nest, pups left scattered and cold, physical abuse of pups, apparent inadequate lactation or feeding.

Oestrous cyclicity (parental animals):

Vaginal lavages were taken early each morning, commencing on the day of pairing, until mating had occurred. The stage of oestrous observed in each vaginal lavage was also recorded.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
- Testes (weighed individually), fixed in Bouin’s fluid
- Epididymides (weighed individually)
- Seminal Vesicles and coagulating gland
- Prostate gland

Litter observations:

The number of live pups born and the number found dead in each litter were recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation and then on Days 7, 14 and 21. The pups were separated by sex and weighed en masse on Days 1, 4, 7 and 14 of lactation. On Day 21 the pups were weighed individually. When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined. Prior to Day 14 of lactation, externally abnormal decedent pups were preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals. Termination of the F0 males was at approximately the same time as the F0 females.
- Maternal animals: All surviving animals. Termination of the F0 females was at or shortly after weaning of their litters (Day 21 of lactation).

GROSS NECROPSY
Necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described in terms of location, size, shape, color, consistency, number and any other relevant characteristics. Representative samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin. The following organs were also fixed:

Ovaries
Uterus (including cervix) and vagina
Testes (weighed individually), fixed in Bouin’s fluid
Epididymides (weighed individually)
Seminal Vesicles and coagulating gland
Prostate gland
Pituitary gland

The reproductive tract of all females was examined for signs of implantation with the number of implantation sites being recorded.

Postmortem examinations (offspring):

SACRIFICE
At terminal necropsy, 2 male and 2 female pups (where they were available) were necropsied.

GROSS NECROPSY
- Gross necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Representative samples of any grossly abnormal tissues were persevered in 10% formalin. The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any abnormal pups found were necropsied as previously described.

Offspring killed or found dead before Day 14 of lactation were checked for the presence of milk in the stomach and the presence of externally visible abnormalities. Any abnormal pups were preserved in 10% formalin or methylated ethyl alcohol as appropriate for possible future examination. Externally normal decedents were discarded.

Offspring killed or found dead on or after Day 14 of lactation were necropsied. This consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of grossly abnormal tissues were preserved in 10% formalin and the carcasses were then discarded.

Statistics:

Body weights and food consumption (for males throughout the study and for females prior to mating) and organ weigh data were analyzed for homogeneity of variance using the “F-Max” test. If the group variances appeared homogenous, a parametric analysis of variance (ANOVA) was used and individual between group comparisons were made using Fisher’s F-protected LSD method via Student’s t-test. If the variances were heterogeneous, log or square root transformations were performed in order to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and a pairwise comparison was made using chi squared protection (via z tests, the nonparametric equivalent of Student’s t-test).
In addition to the ANOVA, organ weights were analyzed by analysis of covariance (ANCOVA) using terminal body weight as covariate. For other parameters, no formal statistical analyses were considered necessary; interpretation of the data being by inspection of the individual and group values.

Reproductive indices:

Fertility Index (male) = Number siring a litter / Number Paired
Fertility Index (female) = Number Pregnant / Number Paired
Gestation Index = Number bearing live pups / Number pregnant
Birth Index = Total number of pups born (live and dead) / Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation / Total number born (live and dead)

Offspring viability indices:

Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0
Lactation Index = Number of pups live on Day 21 of lactation / Number live on Day 4
Overall Survival Index = Number of pups live on Day 21 of lactation / Total number born (live and dead)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical observations that were considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight gains of males throughout the study were comparable with Control. Group mean body weight gains of females prior to mating and throughout gestation and lactation were similar to Control.

Description (incidence and severity):

Group mean food consumption of males throughout the study were comparable with Control. Group mean food consumption of females prior to mating and throughout gestation and lactation were similar to Control.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility and duration of gestation were similar in all groups.

Dose descriptor:

NOEL

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

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Slight intergroup differences in the number of implant sites and survival indices were too small to indicate any association with treatment. Mean litter and pup weights were essentially similar in all groups

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean litter and pup weights were essentially similar in all groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Abnormalities Among Pups
The following abnormalities were observed among pups:
Litter 105 (Group 1): One female pup had a small swelling on ventral abdomen on Day 7.
Litter 126 (Group 2): One pup difficult to sex, suspect female, had no tail or anus evident. Killed on Day 1.
Litter 129 (Group 2): One male pup killed on Day 0 due to gasping, cold to touch, dark in color and unfed.
Litter 137 (Group 2): Days 1-21: One male and one female pup with short, threadlike tails.
Litter 149 (Group 3): Day 0, one female pup killed due to gasping, dark in color and unfed.
Litter 153 (Group 3): One female pup killed on Day 0 due to muzzle being damaged by dam.
Litter 164 (Group 3): Days 4-16 one male pup with swelling on ventral abdomen.
Litter 180 (Group 4): One male pup killed prematurely due to large head and reduced motor function on Day 19.
Litter 184 (Group 4): Day 0: One male pup killed due to gasping, cold to touch, unfed and pale in color. One female pup found dead with right hindlimb not evident. Right fore limb also found to be damaged at pathology external examination.

None of these abnormalities were considered to be attributable to treatment.

Dose descriptor:

NOEL

Generation:

F1

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

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Conclusions:

At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. Mating performance, fertility, duration of gestation, litter size and survival, and litter and pup weights did not indicate any obvious adverse effect of treatment at any of the dose levels tested. Under the conditions of this study, the no observed effect level (NOEL) for both parental and reproductive effects was 1000 mg/kg/day.

Executive summary:

In a One-Generation study according to OECD guideline 415 and in compliance with GLP, the test article was evaluated regarding its effects on general reproductive processes in rats. To that end, the test article was administered to males from 10 weeks prior to mating and to females from two weeks prior to mating, throughout mating, gestation and lactation until the litter had reached Day 21 of lactation. Sprague-Dawley rats were randomized into 3 test groups and 1 vehicle control group, each containing 24 males and 24 females. The animals were dosed once daily by oral gavage at dose levels of 0 (2% medium viscosity CMC containing 0.2% Tween 80), 150, 400, and 1000 mg/kg/day. The dose volume applied was 10 mL/kg body weight. Animals were regularly monitored for clinical signs of toxicity and for body weight and food consumption. The F0 females, along with their F1 pups, were killed shortly after weaning (day 21 of lactation). The males were killed at approximately the same time as the females. All adult (F0) animals were given a gross tissue examination along with 2 male and 2 female F1 pups from each litter. The reproductive organs from the adult (F0) animals were weighed (testes and epididymides only) and fixed. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. Mating performance, fertility, duration of gestation, litter size and survival, and litter and pup weights did not indicate any obvious adverse effect of treatment at any of the dose levels tested. Under the conditions of this study, the no observed effect level (NOEL) for both parental and reproductive effects was 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

To estimate toxicity of the test substance to reproduction, a GLP-compliant one-generation study in Sprague-Dawley rats (OECD guideline 415) was performed. The test article was administered orally by gavage for 10 weeks prior to mating for males and two weeks prior to mating for the females, then throughout mating, gestation and lactation until termination after the litter had reached day 21 of lactation. The animals were dosed once daily by oral gavage at dose levels of 0, 150, 400, and 1000 mg/kg/day. The F0 females, along with their F1 pups, were killed shortly after weaning (day 21 of lactation). The males were killed at approximately the same time as the females. All adult (F0) animals were given a gross tissue examination along with 2 male and 2 female F1 pups from each litter. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. The weights for the male reproductive (testes and epididymides) were essentially similar in all groups. Mating performance, fertility, duration of gestation, litter size and survival, and litter and pup weights did not indicate any obvious adverse effect of treatment at any of the dose levels tested. Some incidental abnormalities were identified among pups in all dose groups, however none of these abnormalities were considered to be attributable to treatment. Therefore, based on the result of this study, the NOEL for parental and filial generation is considered to be 1000 mg/kg body weight per day.

Effects on developmental toxicity

Description of key information

In a prenatal-toxicity-study (OECD 414) 0, 150, 500, and 1000 mg/kg of the test article was given orally by gavage once daily over days 6-19 of gestation to mated rats. The substance did not affect food consumption or body weight gain, also clinical observation and gross necropsy was without any finding. Intergroup differences of pregnancy and fetal weight were not dose dependent and are not considered as treatment related effects. The maternal and fetal no observed effect levels (NOEL) are both considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-28 until 2008-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD®(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 184-293g
- Housing: The animals were housed one per cage in solid-bottomed polypropylene cages (ca 42 x 27 x 20cm); sterilized wood shavings were provided as bedding. A stainless steel food hopper and polycarbonate water bottle were provided for each cage. The cages were suspended on racks.
Cages, bedding and water bottles were changed at regular intervals, as appropriate.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, supplied by Special Diets Services Ltd., Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used during this study is retained in the study archive.
- Water (e.g. ad libitum): The animals had access to domestic quality mains water ad libitum. The supply is analyzed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.
- Acclimation period: The animals were acclimatized in the Charles River animal room from arrival until commencement of treatment on Day 6 of gestation (3-5 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): minimum of 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 h.

IN-LIFE DATES:
Experimental Start Date: 02 May 2008
Experimental Completion Date: 11 July 2008

Environmental Enrichments:
To provide environmental enrichment, wooden chewsticks were made available to the animals, as appropriate. The chewsticks were not considered to contain any additional substances in sufficient concentrations to influence the outcome of the study.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the first 5 days of the study, formulations were prepared daily and used within the established stability period of 24 hours. After 8 days stability had been established, formulations were prepared at approximately weekly intervals, and used within the 8 days stability period (established in Charles River Laboratories Study No. 424799, Method 2479).
An appropriate amount of test item was weighed and placed in a suitably sized glass container, and then the appropriate amount of vehicle was added and mixed by magnetic stirring until a visibly homogenous suspension was obtained.

VEHICLE
The vehicle used was 2% medium viscosity CMC containing 0.2% Tween 80. Full details of the vehicle preparation are retained in the study raw data.

Amount:
10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was undertaken with regard to concentration and homogeneity on two formulation occasions during the study; from formulations prepared for use on Day 1 of the study and Week 2 of the study. Triplicate samples were withdrawn from each formulation, including Control, and were assayed at Charles River, using a method supplied by the Sponsor and previously validated under a separate protocol and contract (Charles River Laboratories Study No. 424799, Method 2479).
The results of the analysis of the dosing formulations were within ±8% of the nominal concentration indicating acceptable accuracy of formulation. The low coefficients of variation (<6.5%) indicated that the formulations were homogenous.

Details on mating procedure:

- Impregnation procedure: cohoused
- Length of cohabitation: 3 successive days
- Proof of pregnancy: day of detection of a vaginal plug or sperm in a vaginal smear = Day 0 of gestation
- further information:
Ninety eight time-mated female rats were obtained in one delivery. The delivery consisted of 3 subbatches, mated over 3 successive days. On delivery, one batch was on Day 1 of gestation, the second on Day 2 and the third on Day 3 (day of detection of a vaginal plug or sperm in a vaginal smear = Day 0 of gestation). No more than two females were mated by any one male.

Duration of treatment / exposure:

over Days 6-19 inclusive of gestation (Day 0 = day of detection of mating).

Frequency of treatment:

animals were dosed once daily, at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24, The spare animals were numbered 97 and 98. These animals were not used and were not regarded as part of the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were agreed with the Sponsor after evaluation of existing relevant toxicological data.
- Animal Identification: Each animal received a subcutaneously implanted electronic identification chip which identified it individually within the study and which corresponded to that animal’s number. It was also given a cage card which was color coded for treatment group and marked with the study number, animal number, cage number and treatment group.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
All the animals were checked for viability early in the morning and again as late as possible each working day throughout the period of the study. In addition, all the animals were examined for reaction to treatment during each day from commencement of dosing. The onset, intensity and duration of any signs were recorded, particular attention being paid to the first 1-2 hours after dosing on each day, then as required thereafter. All the animals were also given a detailed clinical examination, once prior to the start of dosing and daily from commencement of dosing.

BODY WEIGHT: Yes
Body weights were recorded on Days 4 and 6-20 of gestation. For clarity/brevity of reporting, only values on Days 4, 6, 9, 13, 17 and 20 are presented.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The weight of food consumed by each animal was recorded daily, commencing on Day 4 of gestation (weighed quantity first offered on Day 3).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: An external examination was followed by a macroscopic examination of the contents of the thoracic and abdominal cavities; any findings were recorded and representative samples of abnormal tissues were fixed in neutral buffered 10% formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- skeletal examinations: Yes: half per litter
- Head examinations: No data

Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no evidence of maceration), or a late embryonic death (macerated tissue identifiable as an embryo or fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to
distinct, identifiable placenta), or an early embryonic death (discrete, formless, discolored tissue mass attached to the internal uterine wall; may be of varying size).
The foetuses were examined for externally visible abnormalities, each live fetus was individually identified within the litter and its weight was recorded. The fetuses were sexed during subsequent dissection procedures. Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, the remaining half in Bouin’s fluid.
Following initial fixation, the fetuses fixed in alcohol were examined by open dissection for abnormalities of the thoracic and abdominal viscera. These viscera were then discarded. The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification. The fetuses fixed in Bouin’s fluid were examined for soft tissue abnormalities by a technique derived from that of Wilson (1965).

Statistics:

Statistical analysis was performed on body weight gain over Days 6-20 of gestation and on mean fetal weight. Body weight gain was subjected to analysis of variance and fetal weight was analysed by the Kruskal-Wallis test. These tests were two-sided and performed at the 5% significance level. Pairwise comparisons were only performed against the Control group.
For the other parameters, no formal statistical analyses were considered necessary; interpretation of the data being by inspection of the individual and group values.

Historical control data:

The rat is a standard rodent species for the developmental toxicity testing in animals required by the regulatory authorities. The normal processes of gestation in rats and the specific background foetal pathology are well documented in this laboratory.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical observations that were considered to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight gains performance in all treated groups were similar to Control throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption performance in all treated groups were similar to Control throughout the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no necropsy findings that were considered to be related to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on maternal toxic effects:

Intergroup differences in pregnancy performance and foetal weights were negligible due to the slight degree and the absence of a dose-relationship.
At 150 mg/kg/day, a slight increase in the early embryonic deaths was mainly due to Animal 36 that had 13 early deaths; this animal also had a low number of live implants and a low uterus weight. In the context of this study, this isolated finding was not considered to be toxicologically relevant.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The type and distribution of the major foetal abnormalities did not suggest any obvious association with treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The type and distribution of the skeletal abnormalities did not suggest any obvious association with treatment. Slight intergroup differences in the distribution of skeletal ossification parameters were not obviously associated with treatment. At 1000 mg/kg/day, there was a greater incidence of foetuses with incomplete ossification of the thoracic centrum (19 foetuses in 9 litters) and an increase in the number of foetuses with ossified sacrocaudal vertebra with connection between centrum and arches at 150 mg/kg/day (72 foetuses in 20 litters). Although these values were outside the current Control background range of 3 foetuses in 2 litters to 12 foetuses in 6 litters for the first finding and 18 foetuses in 11 litters to 56 foetuses in 17 litters for the latter, the type and distribution of these findings did not indicate any obvious association with treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

The type and distribution of the minor visceral did not suggest any obvious association with treatment.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

From the findings in this study, the maternal and fetal no observed effect level (NOEL) were both considered to be 1000 mg/kg/day.

Executive summary:

In a prenatal development toxicity study in rats (according to GLP and OECD guideline 414), the test item was orally administered to groups of 24 pregnant Sprague-Dawley rats. These animals were dosed by gavage once daily over Days 6-19 of gestation, where the day of detection of mating was designated as Day 0. Dose levels were 0 (2% medium viscosity CMC containing 0.2% Tween 80), 150, 500, and 1000 mg/kg/day. The dose volume applied was 10 mL/kg body weight. Animals were regularly monitored during gestation for clinical signs of toxicity and for body weight and food consumption performance. Animals were killed on Day 20 of gestation. The status of each implantation was recorded and the fetuses were weighed and examined for visceral and skeletal abnormalities, including the state of skeletal ossification. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption performance. Intergroup differences in pregnancy performance and fetal weights were of a slight degree and did not follow a dose-relationship; therefore these variations are not attributable to treatment. The type and distribution of fetal abnormalities and variants did not suggest any obvious effect of treatment. From the findings in this study, the no observed effect level for both maternal and fetal toxicity was considered to be 1000 mg/kg/day.

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

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

To evaluate toxic effects of the test substance on fetal development, a GLP compliant prenatal toxicity study (OECD 414) was performed with pregnant female Sprague-Dawley rats. In the course of the study, 0, 150, 500, and 1000 mg/kg of the test article was given orally by gavage once daily over days 6-19 of gestation to mated female rats. The dose volume applied was 10 ml/kg body weight. Animals were regularly monitored during gestation for clinical signs of toxicity and for body weight and food consumption. At levels up to 1000 mg/kg/day, there were no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. Observed intergroup differences in pregnancy performance and fetal weight were of a slight degree and did not follow any dose-relationship. The type and distribution of major fetal abnormalities and minor visceral and skeletal abnormalities did not suggest any obvious association with treatment. Slight intergroup differences in the distribution of skeletal ossification parameters were not obviously associated with treatment. At 1000 mg/kg/day, there was a greater incidence of fetuses with incomplete ossification of the thoracic centrum and an increase in the number of fetuses with ossified sacrocaudal vertebra with connection between centrum and arches at 150 mg/kg/day. Although these values were outside the current control background range, the type and distribution of these findings did not indicate any obvious association with treatment. Therefore, these variations are not considered as treatment-related effects. From the findings in this study, the maternal and fetal no observed effect levels (NOEL) were both considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, the test substance has not to be classified for reproductive toxicity.