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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09/03/1992-04/04/1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Preliminary Reproduction Toxicity Screening Test (Precursor Protocol of GL 421)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6-bis(1,1-dimethylethyl)-phenol
- Substance type: dialkylphenol
- Physical state: solid at 20°C (colourless solid)
- Analytical purity: 98.7%
- Lot/batch No.: 10/6
- Expiration date of the lot/batch: July 01, 1992
- Instructions for test substance storage: at room temperature in the dark
- Stability in vehicle: 48 hours (reported by the sponsor) (vehicle: bi-distilled water with 4% CMC)
- Safety precautions: goggles and gloves

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd, Wölferstrasse 4, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 11 weeks, minimum
- Weight at study initiation: (P) Males: 304-339 g; Females: 182-229 g
- Fasting period before study:
- Housing: During the prepairing period, males and females were housed separately one to a cage. cages of males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, the rats were housed one male / one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males and females were housed individually again. During the lactation period (until day 4 of lactation), dams were housed together with their litters. Throughout the study, each cage was identified by a colored label for group identification, inscribed with the study schedule number, animal number(s) and details of treatment.
- Diet (e.g. ad libitum): Pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalmuehle AG, CH 4303 Kaiseraugst, Switzerland) was available ad libitum.
- Water (e.g. ad libitum):tap water in bottles was available ad libitum.
- Acclimatization period: 10 days under test conditions, after veterinary examination.
Identification: Individual cage/animal number tattooed on the pinnae


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light. Background music (Schweizerischer Telefonsrundspruch) played at a centrally defined low volume for at least 8 hours during the light period.


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: bi-distilled water with 4% CMC
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (W/v). For preparation of the test article/vehicle mixtures, the test article was melted using a waterbath (40°C). The mixtures were prepared using a homogenizer. During the daily administration period, homegeneity was maintained using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
- M/F ratio per cage: 1 male: 1 female
- Length of cohabitation: maximum of 13 days (except female no. 70 which was paired for 17 days)
- Proof of pregnancy: the day on which spermatozoa were found in the vaginal smear or a vaginal plug was observed was designated day 0 post coitum

- After successful mating each pregnant female was caged (how): animals were separated and housed individually. The females allowed to litter normally and rear their progeny to day 4 of lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1) Concentration, homogeneity and stability of the test article/vehicle mixtures were determined on one occasion prior to the start of the dosing period. samples were taken immediately after preparation and again 2 hours later.
2) During the course of this study, concentration, homogeneity and stability of the test article/vehicle mixtures were determined on two occasions. Analyses were performed by the RCC Analytical Chemistry laboratory using a method supplied by the sponsor.
The samples prepared prior to the start of the dosing period were analyzed using an insufficient analytical method; consequently, results are not reported. The method was improved and validated and analysis was repeated with samples prepared during the dosing period.
Duration of treatment / exposure:
After acclimatization, animals of both sexes received the test article for 14 days prior to pairing and during pairing period (maximum 13 days except female no. 70 which was paired for 17 days). Males continued to be dosed until the day before necropsy. Daily dosing of the females was continued throughout pregnancy and up to day 3 of lactation.
Frequency of treatment:
The test article was administered orally, by gavage, once daily in the morning.
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: (p) minimum 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 150, 750 mg/kg bw/day (P m & f)
Basis:
analytical conc.
10 ml/bw dose, daily dose adjustment to the actual body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were based on the results of a subacute 28-day toxicity study (RCC NOTOX Project 057454).
- Rationale for animal assignment: animals were assigned to the different groups using a computer-generated random algorithm.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health and mortality. Additionally, the females were observed for signs of difficult or prolonged parturition.
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: the animals were weighed daily during the entire study.


FOOD CONSUMPTION :
- Males: food consumption was recorded weekly during the pre-pairing and post-pairing periods but not during the pairing period (mixed values of males and females).
- Females: food consumption was recorded for the following periods: days 1-8 and 8-14 of the prepairing period; days 0-7, 7-14 and 14-21 post coitum and days 0-4 post partum. No food consumption was recorded during the pairing period (mixed values of males and females).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes




OTHER:
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, live birth, stillbirth and any gross anomalies. The sex ratio was recorded. Daily observed for survival and behavioural abnormalities in nesting and nursing.


GROSS EXAMINATION OF DEAD PUPS:
Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: were sacrificed when they were no longer necessary for the assessment of reproductive effects
- Maternal animals: were sacrificed on day 4 post partum.


GROSS NECROPSY
- Necropsy was performed on all animals. At post-mortem examination, all organs were examined and all findings recorded. Samples of the following tissues and organs were collected from all animals at necropsy: testes, ovaries, and all gross lesions.
- The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were rplaced in a solution of ammonium sulfide to visualize possible haemorrhagic areas of implantation sites (Salewski, E., (1964) Arch. Exp. Path. Pharmak. 247, pp. 367-368).


HISTOPATHOLOGY / ORGAN WEIGHTS
The testes and epididymides of all parenteral males were weighed.
The ovaries of all parental females were fixed in neutral phosphate buffered 4% formaldehyde solution.
The testes of all parental males were fixed in Bouin's fixative and embedded in paraffin.
Histological examination was performed on the preserved organs of the highest dose group and the control group. The testes and ovaries were embedded in paraffin wax, sectioned, trimmed, processed at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin and were examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on day 4 post partum.
- These animals were examined macroscopically


GROSS NECROPSY
- Macroscopical


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
The following statistical methods were used to analyse body weights, food consumption, organ weights, reproduction and breeding data:
- If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- For the spontaneous mortality of pups data, Fisher's Exact test 2x2 tables was applied.
Individual values, means, standard deviations and t-statistics were rounded off before printing.
Reproductive indices:
For evaluation of the reproduction data, the following formulae were used:

- pre-implantation loss (%):

number of corpora lutea – number of implantations
------------------------------------------------------------------- x 100
number of corpora lutea


- Post-implantation loss (%):

number of implantations – number of live pups
-------------------------------------------------------------- x 100
number of implantations



- Sex ratio of males/females (%):

number of males/females
---------------------------------- x 100
number of live pups


- Fertility index (%):

number of females achieving pregnancy
------------------------------------------------------ x 100
number of females paired


- Gestation index (%):

number of females with living pups
----------------------------------------------- x 100
number of females pregnant
Offspring viability indices:
- Viability index (%):

number of pups living on day 4 p.p.
----------------------------------------------- x 100
number of living pups at birth

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No deaths occurred during the course of this study in any group, however, severe signs of reaction to treatment were observed in the parent males and females of group 4 (750 mg/kg).
In the parent males, the symptoms were observed from day 2 of the prepairing period until day 5 of the pairing period. During this period the type and intensity was nearly identical after the daily test article administration.
The symptoms mostly observed were ruffled fur, sedation, ataxia, lateral or ventral recumbency, tremor, clonic spasms of the forepaws, hunched posture and salivation.
After day 5 of the pairing period, until termination of the study, the number and intensity of the symptoms diminished continuously and vanished up to ruffled fur in 10 males and salivation in 1 female.
In the parent females, the identical symptoms were noted during the prepairing, pairing and gestation period. During the lactation period, only ruffled fur was observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight males: the calculations of the mean body weight gain resulted in a marginal reduction on the males of group 4 (750 mg/kg) during the prepairing and pairing periods. No further test article-related differences were noted during nay period in any group.
- Food consumption males: In group 4 (750 mg/kg), the mean food consumption was statistically significantly increased from days 8 to 14 of the prepairing period and from days 1 to 10 of the after pairing period. Similar mean food consumption during both periods was noted in group 1 (vehicle control), 2 (3mg/kg) and 3 (150 mg/kg)
- Body weight females: The evaluation of the body weight data resulted in following results:
During the gestation period (days 0-21 post coitum), the mean body weight gain amount to
51.1% in group 1 (vehicle control)
52.8% in group 2 (30 mg/kg)
55.2% in group 3 (150 mg/kg)
47.3% in group 4 (750 mg/kg)
During the lactation period (days 1-4 post partum), calculations resulted in body weight gains of:
8.3% in group 1 (vehicle control)
4.5% in group 2 (30 mg/kg)
6.9% in group 3 (150 mg/kg)
2.8% in group 4 (750 mg/kg)
In group 4, the slight reductions were considered to be test article-related during these two periods. The differences during the prepairing period as well as the differences between groups 1, 2 and 3 during all periods were considered to be incidental.
- Food consumption females: in the females of group 4 (750 mg/kg), the mean food consumption was statistically significantly reduced between days 1 to 8 of the prepairing period. During the second part of the prepairing period (days 8 to 14), the mean food consumption was slightly increased (compensatory reaction). The reduction in group 4 was considered to be test article-related, because in all other groups, the mean food consumption was similar during both periods. No test article-related differences in the mean food consumption of the females were noted during the gestation and lactation periods. The statistically significantly increased mean food consumption in group 3 (150 mg/kg) between days 14 and 21 post coitum isconsidered to be incidental because from the beginning of the study in this group, the highest food consumption was recorded.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- For the mutual reproduction parameters such as precoital time, percentage mating, fertility index and conception rate, there was no indication of test article-related effects in any dose group.
- The evaluation of the primary maternal reproduction data resulted in an increased breeding loss / reduced viability index in the females of group 4 (750 mg/kg). In groups 2 (30 mg/kg) and 3 (150 mg/kg), the data were similar to that in the vehicle control.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The mean organ weights and organ/body weight ratios for testes and epididymides of males were similar in all groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The macrosocpic examination of the parent animals gave no indication of test article-related effects.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The microscopic examination of testes and ovaries of the parent animals of groups 1 (vehicle control) and 4 (750 mg/kg) gave no indication of test article-related effects.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Remarks on result:
other: Generation: P0

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mutual as well as the primary maternal reproduction data were not affected by 2,6-DTBP with exception of an increased breeding loss at 750 mg/kg after day 1 post partum. At this dose level, the body weight gain of pups was reduced from days 1 to 4 post partum (study termination).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mutual as well as the primary maternal reproduction data were not affected by 2,6-DTBP with exception of an increased breeding loss at 750 mg/kg after day 1 post partum. At this dose level, the body weight gain of pups was reduced from days 1 to 4 post partum (study termination).
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)
Birth index:
Group 1: 92.8
Group 2: 83.2 #
Group 3: 95.1
Group 4: 92.1
Viability index:
Group 1: 99.1
Group 2: 99.0
Group 3: 95.9
Group 4: 90.3 ##
#/## : Fischer's Exact test significant at 5% (#) or 1% (##) level.

CLINICAL SIGNS (OFFSPRING)
Postnatal losses: see table in remarks

BODY WEIGHT (OFFSPRING)
Differences in mean body weights and body weight gain: see table in remarks

SEXUAL MATURATION (OFFSPRING)


ORGAN WEIGHTS (OFFSPRING)


GROSS PATHOLOGY (OFFSPRING)
Summary of macroscopical findings F1 pups: see table in remarks

HISTOPATHOLOGY (OFFSPRING)


OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects, exept an increased breeding loss at 750 mg/kg after day 1 post partum

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Summary of postnatal losses during lactation (days 0-4 post partum)

 

Group

Ascertained on day post partum

 

Dead at first litter check

1

2

3

4

1

1F

 

 

 

1F

2

1F

1F

 

 

 

3

 

2F

 

1F

1F

4

 

1M

1M

 

4F, 3M

Differences in mean body weights and body weight gain of F1 pups

 

Group

Days port partum

Body weight gain from day

 

mg/kg

0

g (%)*

1

g (%)*

4

g (%)*

1-4

g (%)**

1

5.5

6.0

8.3

2.3 (+38.3)

2

5.6 (+1.8)

6.0 (±0.0)

8.6 (+3.6)

2.6 (+43.3)

3

5.9 (+7.39

6.4 (+6.7)

9.1 (+9.6)

2.7 (+42.2)

4

5.7 (+3.6)

6.0 (±0.0)

7.4 (-10.8)

1.4 (+23.3)

 

* = percentages refer to the control group

** = alteration within the recording period

Summary of macroscopical findings F1 pups

 

Necropsy of pups found dead at first litter check or died spontaneously during lactation period (day 0-4 post partum)

Group

No. of pups examined

Males/Females

Pup/litter/sex* no(s).

Necropsy day p.p.

Findings

 

1

1

 

 

No abnormal findings

2

1

 

 

No abnormal findings

3

1

 

 

No abnormal findings

4

5

2/73 (M)

3/73 (M)

7/73 (F)

6/78 (M)

4

4

4

1

No milk in the stomach

No milk in the stomach

No milk in the stomach

No milk in the stomach

* = (M) = Male; (F) = Female

 

Scheduled necropsy on day 4 post partum

Group

No. of pups examined

Males/Females

Pup/litter/sex* no(s).

Necropsy day p.p.

Findings

 

1

61/54

9/42 (F)

10/42 (F)

1/44 (M)

6/44 (M)

4

4

4

4

No milk in the stomach

No milk in the stomach

No milk in the stomach

No milk in the stomach

2

52/51

 

 

No abnormal findings

3

50/44

4/61 (M)

6/61 (M)

4

No milk in the stomach

No milk in the stomach

4

43/41

 

4

No abnormal findings

* = (M) = Male; (F) = Female

 

Applicant's summary and conclusion

Conclusions:
The no-observable adverse effect level for the parent animals and pups was considered to be 150 mg/kg body weight/day.
With respect to the reproduction parameters, no effects were noted up to and including 750 mg/kg body weight/day.
No teratogenic effect was observed at external examination and necropsy of the pups in any group.
Executive summary:

A preliminary reproduction toxicity screening study was performed to generate preliminary information concerning the effects of the test material, 2,6 -bis(1,1 -dimethylethyl), on male and female rats reproductive performance such as gonadal functions, mating behaviour, conception and parturition. The method used followed that described in the OECD Guidelines for Testing of Chemicals, March 22, 1990 "Preliminary reproduction Toxicity Screening Test".

The results may be used as a basis for classification and labelling.

The test substance was administered orally, by gavage, to male and female rats (40 males, 40 females, 10 per group) at dose levels of 30, 150, or 750 mg/kg body weight/day. The males received the test article for 43 days and the females up to day 3 post partum.

At 750 mg/kg, administration of test substance caused severe symptoms in male and female rats, increased food consumption in the male rats and reduced food consumption in the female rats. Further, marginally reduced body weight gain in the males and slightly reduced body weight gain in the females were noted.

The mutual as well as the primary maternal reproduction data were not affected by the test article with exception of an increased breeding loss at 750 mg/kg after day 1 post partum. At this dose level, the body weight gain of pups was reduced from days 1 to 4 post partum (study termination).

For all other parameters recorded, no differences were noted between the vehicle control group and any dose group which were considered to be an effect of the test article.

Based on these results, the no-observable adverse effect level for the parent animals and pups was considered to be 150 mg/kg body weight/day.

With respect to the reproduction parameters, no effects were noted up to and including 750 mg/kg body weight/day.

No teratogenic effect was observed at external examination and necropsy of the pups in any group.