2,4,6-tribromophenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tribromophenol

EC Number:

204-278-6

EC Name:

2,4,6-tribromophenol

Cas Number:

118-79-6

Molecular formula:

C6H3Br3O

IUPAC Name:

2,4,6-tribromophenol

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: MANAC, Hiroshima, JAPAN; Lot No.: 70909
- Purity 99.8%
- Storage: cool dark place with appropriate ventilation

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 361-397 g in males, 233-258 g in females
- Fasting period before study: not stated; daily administration of dose
- Housing: individual cages with mesh floor, each cage 6000 cm3; after GD18, dams were housed
in a rearing cage of 14,700 cm3 and additionally given a nursing tray and nesting materials (CareF
RESH). Cages were changed every two weeks.
- Diet: ad libitum with NMF pellets provided by Oriental Yeast Co.
- Water: tap water ad libitum
- Acclimation period: 1-2 weeks (quarantined for one week, study began 2 weeks after arrival)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 2
- Humidity (%): 55 +/-10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The concentrations of dosing solutions were 20, 60, and 200 mg/mL; solutions were made and stored in a tightly closed container in a cool dark place, then used within 8 days.
The test compound was previously determined to be stable for 8 days at concentrations of 20 and 200 mg/mL under the given storage conditions.

Details on mating procedure:

After estrous cycle observation for 14 days before mating females were housed with males of the same group in the same cage, and cohabitated every evening on the basis of 1:1 for 14 days at the longest. Next morning, copulation was confirmed by the presence of vaginal plugs or sperm in the vaginal smear and it was defined as Day 0 of gestation.
Observation of the estrous cycles was continued until successful copulation was confirmed. The number of days from a estrous to the next was designated as the estrous cycle days and the mean estrous cycle was calculated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing solutions were sampled, and confirmed to be within a concentration range of 84.5-100% of the stipulated concentration.

Duration of treatment / exposure:

Total 48 days: Treatment started 14 days prior to mating, and continued daily through the premating period, mating, gestation, and until lactation day 3.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

As acute oral LD 50 was previously reported as 2000 mg/kg bw., a preliminary test of 14 days was conducted with the doses of 0 (vehicle; corn oil), 100, 300 and 1000 mg/kg/day.
Based on the results of the preliminary test, the highest dose was set at 1000 mg/kg/day where clear adverse effects would appear.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes, Clinical observations were conducted for all animals of both sexes every day throughout the study period, and abnormalities and deaths noted.
BODY WEIGHT: Yes; Body weights and food consumption of males were measured prior to dosing, then on Days 1, 8, 15, 22, 29, 36, 43, and 48 (food) 49 (weight; necropsy date).
Body weights and food consumption of females were measured prior to dosing, then Days 1, 8, and 15. Non-pregnant females were measured Days 22, 29, 36, 43, and 49. Pregnant females were measured on gestation days 0, 7, 14, 21, and lactation days 0 and 4.
FOOD CONSUMPTION
Food consumption was measured on Day 1 (the date of starting administration), 8 and 15 of administration. The food consumption from the measurement day to the next measurement day was obtained to calculate the mean daily food consumption and cumulative food consumption from Day 1 to 15 of administration. For females with unsuccessful copulation, food consumption was measured on Day 29, 36, 43 and 48 of administration after the failure of copulation to calculate food consumption from the measurement day to the next measurement day. Based on these, the mean daily food consumption was calculated. For females with successful copulation, and females which delivered pups, food consumption was measured on Day 0, 7, 14 and 21 of gestation, on Day 0 and 4 of lactation respectively, and the food consumption was calculated from the measurement day and to the next measurement day. The mean daily food consumption and the cumulative food consumption from Day 0 to 21 of gestation were calculated.
No measurement of food consumption was conducted in the cohabitated animals during the mating period.
CLINICAL CHEMISTRY: Yes
- Parameters checked were examined: total protein, albumin, A/G, ALP, Creatinine, total bilirubin, potassium, chloride.

OTHER:
Observation of newborns at the spontaneous delivery.
All the dams were allowed to make spontaneous delivery. Confirmation of delivery was conducted 9 - 10 AM. If completion of delivery at this time zone was confirmed the day was defined as Day 0 of lactation. For animals started parturition in this time zone, completion of parturition was allowed and the day was defined as Day 0 of lactation. If parturition was completed after 10 AM, the next day was defined as the Day 0 of lactation. The dams were subjected to pathological necropsy on Day 4 of lactation.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored for 14 days before mating, which took place 1:1 with males every evening for up to 14 days; the morning a vaginal plug or sperm was detected in a vaginal smear, the female was defined as Day 0 of gestation.
Dams were allowed to deliver spontaneously. If delivery was complete by 10 am, the day was defined as Day 0 of lactation. If parturition continued after 10 am, the following day was designated Day 0 of lactation.

Litter observations:

On Day 4 of lactation, all the newborns were subjected to necropsy to conduct macroscopic observation of organs and tissues. The macroscopic observation of organs and tissues were conducted in the similar manners also for pups which died during the lactation period.

Postmortem examinations (parental animals):

Sacrifice and pathology:
On lactation day 4, all dams and pups were sacrificed and necropsied. Pups that died before lactation day 4 were also subjected to necropsy.

GROSS PATHOLOGY: Yes, liver, kidney, thymus, brain, adrenals testes

HISTOPATHOLOGY: Yes
Clinical laboratory tests were performed in all males of each group at necropsy. The males were fasted for 16 hours, then anaesthetized with ether, and blood collected from the abdominal aorta for haematology testing.

Statistics:

Multiple comparison assay for body weight, for food consumption, for numbers of corpora lutea, implantation sites, pups born, and stillborns, for sex ratio, for mean estrous cycle, for gestation period, for indices of implantation, delivery, viability on lactation day 4, and live births, for incidence of external anomaly, for organ weight and ratio of organ and body weights, and for haematological and blood chemistry values. Bartlett's test for variance was initially performed , followed by a one-way analysis
of variance if variance was similar within groups. If variance was unequal within groups, Kruskal-Wallis rank test was done, followed by Dunnett's multiple comparative assay (non-parametric). The Chi-squared test was used for indices of delivery, copulation, and fertility. Fisher's direct probability test was used for incidence of pathological results, and the Mann-Whitney U test used to examine the rank sum. Results were noted as significant at p<0.05 or p<0.01.

Reproductive indices:

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: Number of corpora lutea, Number of implantations

Offspring viability indices:

Based on the mating result, copulation index [(the number of animals with successful copulation/the number of cohabitated animals) x 100] was calculated for each group.
For all the cases in which delivery was confirmed, calculation was made for the gestation period (the number of days which was obtained by reducing the dates of Day 0 of lactation from the date of Day 0 of gestation), the fertility index [(the number of animals with conceptus / the number of copulated animals) x 100], gestation index [(the number of dams delivered live pups / the number of pregnant females) x 100], implantation index [(the number of implantation site / the number of corpora lutea) x 100], delivery index [(the total number of pups / the number of implantation site) x 100], live birth index [(the number of live pups / the total number of pups) x 100].

The viability index on Day 4 of the newborns [(the number of live pups on Day 4 of lactation/the total number of pups born) x 100] was calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in both males and females within 30 minutes of dosing at 300 and 1000 mg/kg. Numbers of animals with salivation were increasing by increased dose. This salivation, therefore, was considered as an adverse effect caused by the administration of this chemical. At 100 mg/kg/day no abnormality was observed.
Other signs were sporadic across treatment groups.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed for all groups up to the highest doses, 1000 mg/kg/day for the test period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males dosed at 1000 mg/kg had statistically lower body weights compared to controls beginning on Day 8 of the study. Male body weight gain at 1000 mg/kg was also significantly low. Females did not show any difference in body weight or body weight gain until after Day 7 of gestation at the 1000 mg/kg dose level. Body weight and body weight gain were both low in the 1000 mg/kg group to Day 4 of lactation (date of sacrifice).
At 300 and 100 mg/kg/day: No significant changes were observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In males, the 1000 mg/kg group had significantly lower food consumption compared to controls from Day 1 to 8 of the study. In females, the 1000 mg/kg group also had significantly lower values from Day 1 to 8 in the premating period, but also from Day 0 to 4 of lactation. No differences were noted during the gestation period.
In the 300 and 100 mg/kg/day dose groups no significant changes were observed.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There was not considered to be a significant difference between treatment groups and contols, although the blood coagulation test showed a slight but statistically significant prolongation time compared to controls.
At 100 to 1000 mg/kg/day: No adverse effects were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically elevated creatinine levels were seen in the 300 and 1000 mg/kg dose groups compared to controls. In 1000 mg/kg groups only, elevated protein was seen in total protein, albumin, A/G, ALP, and chlorine, as well as a higher trend in BUN, and significantly lower values of total bilirubin and potassium.
At 300 mg/kg/day or more: Significant increase in creatinine was observed. This increase suggested the toxicity of this chemical to liver and kidneys.
At 100 mg/kg/day: No significant adverse effects were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Pups in one litter treated with 1000 mg/kg all died after birth; the dam of this litter showed pigment deposits in the spleen, cellular infiltration in the lung, fatty changes in the liver, and microgranuloma in the uterus.
In males treated with 1000 mg/kg, changes seen were atrophy of the thymus (3 animals), hypertrophy of hepatocytes (12 animals), hyaline cast in kidney (8), dilatation of kidney tubles (7), renal papillary necrosis (5), lumphocyte infiltration (6), resulting in an increases incidence of observations compared to control. Fatty changes in liver and basophilic tubules were noted as mild in other treatment groups, but the degree of severity was greater in the 1000 mg/kg group. Elevated incidence of eosinophilic bodies were seen in the kidney of the 100 mg/kg group, but there was no dose-response or increase in severity. Pigment deposits in spleen, hemorrhage in thymus, microgranuloma in liver, and vacuolar degenertaion in adrenal zona fasciculata were observed frequently across groups in
cluding control. Othe sporadic findings were seen but not specified.
In females, the observed changes were either sporadic or without difference in incidence among groups.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

HISTORICAL CONTROL DATA (if applicable): not stated

OTHER DATA:
In animals which failed to copulate, pigment deposits and lymphocyte infiltration were seen in the kidneys of both sexes. The male additionally had lymphocyte infiltration in liver, basophilic tubules in the kidney, and angiectasis and vacuolar degeneration of the zona fasciculata in the adrenals. The female additionally had capsulitis in the spleen, pneumonia, microgranulation in the liver, pyelitis in kidney, dilatation of the lumen and cellular infiltration of the uterus.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Almost 4 - 5 days of estrous cycle was noted in all groups in the observation of the estrous cycles, no inter group difference was noted in the mean estrous cycles.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- COPULATION AND FERTILITY
Copulation was achieved in all animals of the groups treated with the test substance except the control group. In the control group, one pair failed to achieve copulation and the copulation index was 91.7%. Conception was established in copulated females of all groups. Almost 4 - 5 days of estrous cycle was noted in all groups in the observation of the estrous cycles, no inter group difference was noted in the mean estrous cycles.

- DELIVERY AND LACTATION
Statistically significantly low values were noted in gestation period in 1000 mg/kg group compared to the control group. However, individual gestation period was 22 days in all animals, indicating that they were within the normal ranges. The number of live pups on Day 4 of lactation was significantly low in both sexes in the same group compared to the control group, viability index on Day 4 after birth was also significantly low in both males and females.
No abnormalities were noted in delivery conditions, the number of corpora lutea, number of implantation site, number of newborns and stillborns were almost the similar values, indicating no inter-group differences in the gestation index, implantation index, delivery index and live birth index.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg group viability index on Day 4 after birth was significantly lower in both males and females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight during the lactation period was statistically significantly lower on Day 0 and 4 after birth in males and females of the 1000 mg/kg group compared to the control group.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Details on embryotoxic / teratogenic effects:
Morphological findings of newborns, body weight and necropsy findings:
In external investigation of newborns, imperforate anus and absence tail were observed in a newborn in 100mg/kg group, imperforate anus and rudimentary tail were observed in a newborn in 300 mg/kg group.
Concerning the body weight during the lactation period, statistically significantly low values were noted on Day 0 and 4 after birth in males and females of 1000 mg/kg group compared to the control group. In the necropsy of dead pups, abnormality at the origin of right subclavian artery was observed in 1 and 3 pups of 100 and 1000 mg/kg group, respectively. Enlargement of renal pelvis was observed in a pup of 100 mg/kg group, hypoplastic brain and anomaly of esophagus location in a pup of 300 mg/kg group, small eye balls in a pup of 1000 mg/kg group, respectively. In the necropsy on Day 4 of lactation, thymic remnant in the neck was observed in 3, 2 and 1 males in the control group, 100 and 300 mg/kg group, respectively. White patch of liver was observed in a pup of the control group and 1000 mg/kg group, respectively. Red patch of liver, black patch and pale color of liver were noted in 1 to 2 pups of 300 or 1000 mg/kg group.
In females, thymic remnant in the neck was observed in 2, 1 and 1 pups of the control group, 100 and 1000 mg/kg group, respectively. White patch of liver were observed in 1 and 4 pups of 100 and 1000 mg/kg groups and significantly high incidence was noted while in 1000 mg/kg group compared to the control group. Red patch, black patch and green color of liver, imperforate anus, red eye balls, absence tail, rudimentary tail and crust were sporadically observed in the groups treated with the test substance.

All liveborn pups of a dam in 1000 mg/kg group was noted on Day 4 of lactation (animal No. 2306).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

For the reproductive testing, no effects were seen on the number of pairs mated, the pairs copulated nor the number of pregnant females, the mean estrous cycle, copulation, or fertility. No obvious effect of treatment was noticed during gestation, and deliveries were normal. No effect was seen on number of corpora lutea, implantation sites, number of pups born or live-born, the implantation index, or the delivery index.

Applicant's summary and conclusion

Conclusions:

No adverse effects were observed in estrus cycle, copulation, fertility results and duration of gestation period as well as finding for delivery, number of corpora lutea, implants, total pups and live pups born and implantation and delivery indices in any treatment groups.
With regard to the effects on neonates, viability on day 4 lactation and body weights on day 0 and 4 of lactation in 1000 mg/kg/day group were lowered in both sexes.
In conclusion, NOAELs for reproductive/developmental toxicity are considered to be 1000 mg/kg/day for parents and 300 mg/kg/day for the F1 generation. But because of maternal toxicity P0 at 100 mg/kg/day the overall NOAEL for 2,4,6-tribromophenol in this adequate repeated-dose study is therefore 100 mg/kg/day in both sexes. The overall NOAEL is considered protective of reproduction and development because it is lower than the level at which effects are seen for reproduction and development.