2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)

Toxicity to reproduction

Administrative data

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:

1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1969

Report date:

1969

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1966

Report date:

1967

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Principles of method if other than guideline:

The general reproductive performance of the mice was investigated after a total of 36 weeks of treatment. To that end, a reproductive study covering two generations was introduced. Mice within each of the two groups were mated at 36 weeks from commencement of feeding the test material. At 28 days post partum the weanlings were reduced, by random selection, to totals of 28 males and 28 females per group. The F1 generation was reared to three months of age, while being caged in fours and fed control or test diet as appropriate. Mating and reproductive performance were then studied exactly as in the case of the F0 generation.

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)

EC Number:

230-426-4

EC Name:

2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)

Cas Number:

7128-64-5

Molecular formula:

C26H26N2O2S

IUPAC Name:

5-tert-butyl-2-[5-(5-tert-butyl-1,3-benzoxazol-2-yl)thiophen-2-yl]-1,3-benzoxazole

Test animals

Species:

mouse

Strain:

other: hysterectomy-derived strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, U.S.A.
- Age at study initiation: (P) 36 wks; (F1) 12 wks (start: mating and reproductive performance)
- Weight at study initiation: (P) Males: 28-37 g (31 g group mean bw); Females: 21-28 g (23 g group mean bw); (F1) Males: 16-36 g (26 g group mean bw); Females: 20-31 g (24 g group mean bw)
- Housing: polypropylene cages (North Kent Plastics, Limited) four to a cage, except where the number was reduced during the mating phase.
- Diet: Sifted Spiller's Laboratory Small Animals Diet (autoclaved), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data.
- Humidity (%): No data.
- Air changes (per hr): No data.
- Photoperiod (hrs dark / hrs light): No data.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: vehicle: feed

Details on exposure:

Not further specified.

Details on mating procedure:

- Mating: 36 weeks from commencement of feeding the test substance.
- M/F ratio per cage: one male to one female.
- Length of cohabitation: males were returned to their original cages three days prior to the anticipated parturition date.
- Proof of pregnancy: Sperm in vaginal smear or copulatory plug referred to as day 0 of pregnancy (female mice were examined twice daily)
- Replacement of first male by another male with proven fertility after unsuccessful pairing (days): not noticed
- Further matings after two unsuccessful attempts: not noticed
- After successful mating each pregnant female was caged: individually (males were returned to their original cages)
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

36 weeks prior to mating until sacrifice

Frequency of treatment:

daily (in diet)

Details on study schedule:

- F1 parental animals not mated until 8 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 12 weeks (3 months)
- Mating and reproductive performance: studied exactly as in the case of the F0 generation.
- F1 generation:
# All young that died were autopsied in an attempt to determine the cause of death.
# At 28 days post-partum the weanlings were reduced to totals of 28 males and 28 females per group; young killed at this time were subjected to gross examination.

Doses / concentrations

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

52

Control animals:

yes, plain diet

Positive control:

None.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/MORTALITY: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weighed initially, then at weekly intervals for the first 26 weeks, every two weeks to 52 weeks, and thereafter at four-weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Several checks on consumption were made over monthly periods at intervals throughout the investigation.

REPRODUCTION:
- Mating performance
- Conception rate (ratio of the number of litters born to the number of females paired, expressed as a percentage)
- Gestation period (interval between the occurrence of a positive smear (or a copulatory plug) and parturition)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- As soon as possible after birth (12 hours), all young were counted and examined for external abnormalities. At 24 hours, all young were weighed and identified by toe amputation, and the ano-genital distance was measured. All litters were examined daily for dead young (which were autopsied), and weighed at weekly intervals up to 28 days postpartum. At all times, nest disturbance was kept to a minimum.
- numerical litter sizes at birth and at weaning (mean values were calculated after exclusion of data from litters in which total perinatal mortality was recorded)
- litter weights and mean pup weights, at birth and at weaning (calculated from individual pup weights)
- number and sex of pups
- stillbirths
- live births
- postnatal mortality (pup mortality); percentage losses to weaning ([total number of pups born - total number weaned] / total number of pups born) x 100
- presence of gross anomalies (12 h after birth)
- weight gain (weekly intervals up to 28 days postpartum)
- physical or behavioural abnormalities
- other: ano-genital distance (24 h after birth)
- other:
# At 28 days the pups were weaned; 28 males and 28 females per group were randomly selected to survive to maturity .
# The surplus young were sacrificed at this time and examined externally and internally for abnormalities; sex was determined by gonadal inspection.

The following parameters were examined in F2 offspring:
- bodyweight and food consumption

GROSS EXAMINATION OF DEAD PUPS: yes
- for external and internal abnormalities
- possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- All mice found dead, or sacrificed in extremis, were subjected to detailed macroscopic examination to locate the affected organ(s) and, if possible, to ascertain the cause of death. Where practicable, a full spectrum of tissue-samples was preserved in formaldehyde saline.
- All mice surviving to termination were sacrificed by carbon dioxide euthanasia. The macroscopic appearance of the tissues was noted and the weights of liver and kidneys recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated below were prepared for microscopic examination: adrenals, caecum, colon, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, rectum, spleen, stomach, all abnormal growths

- In the first instance microscopic examination of the tissues was confined to:
(i) abnormal growths from all mice,
(ii) at least 10 males and 10 females surviving to termination in each group.
This was subsequently extended to include all mice dying after week 36.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: abnormal growths from all mice in which they occurred

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (F0, F1, F2 generation)

MICROSCOPIC EXAMINATION:
- F1 generation: 5 males and 5 females from each group
- F2 generation: 5 males and 5 females from each group

HISTOPATHOLOGY
The tissues indicated as for the F0 generation were prepared for microscopic examination.

Reproductive indices:

Mating performance, Reproductive performance, Gestation period

Offspring viability indices:

Percentage live males at first litter check, Percentage live females at first litter check, Percentage of postnatal loss days 0 - 4 of lactation, viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no overt signs of reaction to treatment amongst either the F0 mice or the later generations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality rates and prime causes throughout the investigation remained grossly comparable for the two groups. Three mice from the F0 control group were sacrificed during the reproductive phase on account of dystocia. The females of the F0 treated group survived parturition without loss, as did all females of the F1 generation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean bodyweights for the treated F0 females remained slightly, but consistently, below those of the control females. The difference was not progressive, although it was maintained in spite of increased food consumption in testing Group 2, Viewed in the light of growth observations in succeeding generations, it was not considered to relate to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

An increase in food intake, equivalent to 2g/mouse/week, was maintained by the female test mice throughout the treatment period and continued when the ratce were fed control diet. Food intakes for the two male groups remained comparable throughout the investigation.
No distinct pattern emerges from these data, and it was concluded that treatment exerted no significant effect on food consumption.

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In respect of non-neoplastic histology, there were no indications of any treatment-related differences between the treated and control mice.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Very few tumours were seen in mice of the F1 and F2 generations. The table below contains data for the F0 generation only. Among the mice of the F0 generation, liver tumours were observed in 9 out of 52 control males, whilst in the treated males the incidence was 14 out of 52. These incidence data lie within the normal range for this strain of mouse. Only three tumours were seen in mice of the F1 generation; these comprised two reticuloendothelial tumours (both in control mice that died before termination) and one lung tumour (in a treated male sacrificed at termination). One reticulo-endothelial tumour was found in a control male of the F2 generation.
There was, therefore, no evidence to suggest that the test substance, at 1000 ppm in the diet, might be carcinogenic for mice.

Description (incidence and severity):

Prior to sacrifice, a representative sample of mice from each generation was examined under ultraviolet light.
All treated mice showed evidence of localized deposition. In the intact mouse, fluorescing areas included the skin, base of the pinnae, tail and limbs.
At autopsy it was evident that deposition had occurred primarily in the adipose tissue; the fluorescence seen in the intact animal was apparently due to the location of this tissue. The degree of fluorescence was much greater in mice of the F1and F2 generation (which were still being maintained on treatment) than in the F0 generation mice (which had been withdrawn from treatment for 26 weeks prior to sacrifice). Throughout all generations the intensity of fluorescence in females was greater than in males, presumably due to the differing amounts of adipose tissue deposited in the two sexes.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

MATING PERFORMANCE:
In the F0 generations, the majority of mice that mated did so at the first available oestrus. A number of mice showed evidence of an ineffectual mating.
Although a plug was seen, parturition occurred 24-25 days later, indicating that fertilization had occurred only after mating at the next oestrus; this in all probability was a result of the so-called "strange male" effect (Bruce, 1959: Nature, 184, 105) observed in 4+4 mice (control and treated, respectively) of the F0 generation. Although this suggested an apparent increase in the number of such ineffectual matings in the F1test group, the difference is not statistically significant.

CONCEPTION RATE:
The performance of the F0 treated mice was slightly superior to that of the control group.

GESTATION PERIOD:
In the F0 generations, the two groups showed closely comparable mean gestation times. The dispersion of the individual values for each group (Chi-squared test) showed no significant difference.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive performance of the parent mice was unaffected at a dosage of 1000 ppm (formally equivalent to 150 mg/kg bw/d).

Results: P1 (second parental generation)

General toxicity (P1)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- F1 generation: In the 34 weeks following weaning, both sexes gained more weight than did their respective controls. This increase correlated with increased food consumption in testing Group 2.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- F1 generation: Both sexes of testing Group 2 showed an increase in food consumption equivalent to 1 g/mouse/week.

Reproductive function / performance (P1)

Reproductive performance:

no effects observed

Description (incidence and severity):

MATING PERFORMANCE:
In F1 generations, the majority of mice that mated did so at the first available oestrus. A number of mice showed evidence of an ineffectual mating. Although a plug was seen, parturition occurred 24-25 days later, indicating that fertilization had occurred only after mating at the next oestrus; this in all probability was a result of the so-called "strange male" effect (Bruce, 1959: Nature, 184, 105) in 1+5 mice (control and treated, respectively) of the F1 generation. Although this suggested an apparent increase in the number of such ineffectual matings in the F1test group, the difference is not statistically significant.

CONCEPTION RATE:
The performance of the F1 treated mice was slightly lower to that of the control group.

GESTATION PERIOD:
In F1 generations the two groups showed closely comparable mean gestation times. The dispersion of the individual values for each group (Chi-squared test) showed no significant difference.

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

NUMERICAL LITTER SIZE:
At birth, litter sizes were comparable for both groups. At weaning, mean litter size for treated mice was not significantly different from that of controls.

PUP MORTALITY:
The slightly higher losses during lactation in the treated group were counterbalanced by the fact that a greater proportion of control pups died in litters lost in toto: overall pup losses, therefore, were essentially the same in both groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

LITTER WEIGHT AND AND MEAN PUP WEIGHT:
At birth and weaning, litter weight for treated mice appeared slightly lower than that of the control mice; however, this difference was not statistically significant. Conversely, mean pup weight of the treated mice was higher than that of the control mice, possibly reflecting the reduced competition arising from the lower litter size.

Description (incidence and severity):

ANO-GENITAL DISTANCE:
Retrospective comparison with organic sex (as determined by gonadal inspection at autopsy) showed that, in both generations of both groups, female offspring had ano-genital distances (AGD) at birth not exceeding 2.5 mm; pups with AGD in excess of 3.5 mm were male, while both sexes were equally represented amongst young with AGD in the range 2.8 to 3.2 mm. In both reproductive cycles of this study, neither AGD distribution at birth, nor the sex ratio at weaning, was affected by treatment with the test substance.

Description (incidence and severity):

No morphological abnormalities were detected at any stage during this experiment, either among stillborn pups or in pups that died during the lactating period.

Details on results (F1)

MATING PERFORMANCE:
In F1 generations, the majority of mice that mated did so at the first available oestrus. A number of mice showed evidence of an ineffectual mating. Although a plug was seen, parturition occurred 24-25 days later, indicating that fertilization had occurred only after mating at the next oestrus; this in all probability was a result of the so-called "strange male" effect (Bruce, 1959: Nature, 184, 105) in 1+5 mice (control and treated, respectively) of the F1 generation. Although this suggested an apparent increase in the number of such ineffectual matings in the F1test group, the difference is not statistically significant.

CONCEPTION RATE:
The performance of the F1 treated mice was slightly lower to that of the control group.

GESTATION PERIOD:
In F1 generations the two groups showed closely comparable mean gestation times. The dispersion of the individual values for each group (Chi-squared test) showed no significant difference.

LITTER DATA:
Based on the observations of the breeding performance, effects on the separate litter parameters are discussed below.

NUMERICAL LITTER SIZE:
- F0 generation: At birth, litter sizes were comparable for both groups. At weaning, mean litter size for treated mice was not significantly different from that of controls.
- F1 generation: At birth and weaning, litter sizes were grossly comparable for both groups.

LITTER WEIGHT AND AND MEAN PUP WEIGHT:
- F0 generation: At birth and weaning, litter weight for treated mice appeared slightly lower than that of the control mice; however, this difference was not statistically significant. Conversely, mean pup weight of the treated mice was higher than that of the control mice, possibly reflecting the reduced competition arising from the lower litter size.
- F1 generation: At birth and weaning, the relationship seen in the F0 generation mice was reversed; mean litter weight in Group 2 was slightly, but not significantly, higher than in the control group. The small intergroup difference in mean pup weight as in the F0 generation, reflected the effect of within litter competition.

ANO-GENITAL DISTANCE:
Retrospective comparison with organic sex (as determined by gonadal inspection at autopsy) showed that, in both generations of both groups, female offspring had ano-genital distances (AGD) at birth not exceeding 2.5 mm; pups with AGD in excess of 3.5 mm were male, while both sexes were equally represented amongst young with AGD in the range 2.8 to 3.2 mm. In both reproductive cycles of this study, neither AGD distribution at birth, nor the sex ratio at weaning, was affected by treatment with the test subsatnce.

PUP MORTALITY:
- F0 generation: The slightly higher losses during lactation in the treated group were counterbalanced by the fact that a greater proportion of control pups died in litters lost in toto: overall pup losses, therefore, were essentially the same in both groups.
- F1 generation: Pup losses during the lactating period were lower than in the preceding generation and were alnnost identical in the two groups. Overall pup losses were again essentially the same in both groups.

ABNORMALITIES:
No morphological abnormalities were detected at any stage during this experiment, either among stillborn pups or in pups that died during the lactating period.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive performance of F1 generation was unaffected at a dosage of 1000 ppm (formally equivalent to 150 mg/kg bw/d).

Results: F2 generation

General toxicity (F2)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

NUMERICAL LITTER SIZE:
At birth and weaning, litter sizes were grossly comparable for both groups.

PUP MORTALITY:
Pup losses during the lactating period were lower than in the preceding generation and were alnnost identical in the two groups. Overall pup losses were again essentially the same in both groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The growth rates of treated and control mice were grossly comparable despite decreased food consumption in Group 2 (females).

LITTER WEIGHT AND AND MEAN PUP WEIGHT:
At birth and weaning, the relationship seen in the F0 generation mice was reversed; mean litter weight in Group 2 was slightly, but not significantly, higher than in the control group. The small intergroup difference in mean pup weight as in the F0 generation, reflected the effect of within litter competition.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treated females consumed less than controls, by an amount equivalent to 2g/mouse/week. The two male groups consumed identical amounts of food during a four week recording period.

Description (incidence and severity):

No morphological abnormalities were detected at any stage during this experiment, either among stillborn pups or in pups that died during the lactating period.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

The general reproductive performance of the mice was investigated after a total of 36 weeks of treatment. Treatment continued throughout the mating, gestation and lactation periods. The F1 generation was reared to maturity and mated; treatment continued throughout this time, as for the F2 generation. The progeny of F1 generation were reared to maturity under treatment and then sacrificed. Reproductive performance of the parent mice and F1 generation was unaffected, at all stages, by treatment with the test article. No significant effect of treatment was discernible in overall consideration of the litter parameters. No morphological abnormalities were recorded at any stage of the reproductive studies. Throughout the investigation, mortality rates and prime causes remained grossly comparable for the treated and untreated groups. No treatment-related trends were seen in growth or food consumption. Examination of the treated mice under UV illumination revealed fluorescent deposits in adipose tissue. After 26 weeks withdrawal from treatment, fluorescence was somewhat reduced, although still evident. Tumour incidence was unaffected by treatment. It was concluded that there was no evidence of adverse reaction to treatment, and that deposition of fluorescent material constituted the sole treatment-related finding.