2,4,6-tribromophenol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated; published 1998

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The exposure was continuous whole-body inhalation chamber treatment for 21 days, so there was unavoidable oral exposure from grooming. The doses are therefore nominal not actual. Additionally, no analysis was mentioned to validate the nominal dose. Original data is not given, results are mentioned in the text or given as figures. Results are qualitatively valuable only.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

25 pregnant Wistar rats per treatment group were exposed to 2,4,6-tribromophenol by whole-body inhalation from day 1 to day 21 of gestation for 24 hours/day, 7 days/week. Groups were housed together in an inhalation chamber and exposed to a fixed concentration of test compound. The effects of the test compound were monitored by assessing skin pain threshold (method of Speranskiy, 1965), and by assessing behavior in a modified open field (Martson and Shepelskaya, 1980). These tests were performed on dams at gestational day 21, and on the pups on postnatal days 30 and 60.

GLP compliance:

not specified

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

Details on test material:

The only details stated are that the test material was of research grade, and obtained from a local manufacturer, NPO "Iodobrom", Saci, Russia.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals were obtained from Rappolovo animal facitity, St Petersburg, Russia

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Remarks:

physical form and preparation of test compound for inhalation are not described

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: specially designed chamber, description not available
- Method of holding animals in test chamber: n/a, whole body exposure
- Source and rate of air: not defined
- Method of conditioning air: not described
- System of generating particulates/aerosols: not described
- Temperature, humidity, pressure in air chamber: 21+/- 2 degrees C, relative humidity of 55 +/- 15%
- Air flow rate: not described
- Air change rate: 12 changes per hour
- Method of particle size determination: not described, no statement that particle size or doses were analyzed
- Treatment of exhaust air: not described

TEST ATMOSPHERE
- Brief description of analytical method used: none described
- Samples taken from breathing zone: not described

VEHICLE (if applicable): not described, assumed not applicable

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information

Details on mating procedure:

Three females of size 180-200 g were housed with one male for mating. There is no description of the male, and no statement that the male was of proven fertility. Day 1 of pregnancy was determined as the day of finding spermatozoa in vaginal smears.

Duration of treatment / exposure:

Pregnant dams were exposed continuously from Day 1 to Day 21 of gestation in a whole-body chamber with 24 other dams. There is no statement regarding oral exposure from grooming, or variability in exposure from filtering particles.

Frequency of treatment:

Continuously

Duration of test:

21 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.03, 0.1, 0.3, 1.0 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

25 dams per dose, no exposure of males used for mating

Control animals:

yes, sham-exposed

Details on study design:

None

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: not stated

FOOD CONSUMPTION AND COMPOUND INTAKE: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data


POST-MORTEM EXAMINATIONS: No data

OTHER: At undefined time(s), maternal body weight, rectal temperature, lipid peroxidation in liver and placenta, level of total amino nitrogen in urine and blood, excretion of phenols in urine, progesterone, estradiol and corticosterone in blood plasma, alkaline phosphatase, peroxidase and catalase blood levels, phagocytotic index, and blood antimicrobial activity were monitored. The influence of tribromophenol on the central nervous system was monitored by assessing skin pain threshold, using the method of Speranskiy (1965) and modified by Pavlenko and Guseva (1975) and by observing behavior reactions in a modified open field device.

Ovaries and uterine content:

Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- 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: Yes [assume all]
- Number of live and dead fetuses
- Examined for visceral abnormalities
- Behavioral observations (age at which both eyes were opened, first day of bilateral appearance of upper and lower incisors, behavior reactions in a modified open field device (Speranskiy 1965) on postnatal day 30 and 60 were determined)
- Relative weights of heart, liver, kidneys, spleen, adrenal gland, testis (or ovary)

Statistics:

Statistical analysis was performed using SAS software version 6.12. For continuous outcome variables one-way ANOVA was used (PROC GLM), and Dunnett's two-tailed t-test was used to identify the concentrations different from control values. Wilcoxon rank-sum test was applied to incidence of visceral and skeletal variants.

Indices:

Not stated

Historical control data:

Not stated

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: unclear whether the listed effect on open field behavior is adverse

Details on maternal toxic effects:
On gestation day 21, dams were observed in an open field and quantitated for movement-related effects. A significant decrease in nose-poking behavior (described as orientation reaction) was noted at 1 mg/m3, p<0.05. Also at this exposure level, increases in the level of alkaline phosphatase in blood, total amino nitrogen in urine, excretion of total phenols in urine, and level of progesterone in blood were seen. There were no effects at lower exposures. Caveat: this is a whole-body exposure study and grooming behavior would produce additional oral exposure.
There were no effects on physiological parameters such as nonspecific immunological function, body weight and temperature, and no effect on other behavioral measures such as horizontal and vertical movement, latency for movement, and pain threshold.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEC

Effect level:

1 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEC

Effect level:

0.3 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

NOEC

Effect level:

< 0.03 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: developmental toxicity

4

Dose descriptor:

NOEC

Effect level:

ca. 0.03 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A significant increase was noted in total embryolethality, seen as the difference between numbers of corpora lutea and live pups at gestation day 15. Preimplantation and postimplantation loss was shown. The lowest dose tested, 0.1 mg/m3, produced a significant effect; note however that the whole body exposure is likely to have produced oral exposure that would have added to the inhalation dose.
Hematomas were increased in the 1.0 mg/m3 group (0.31 vs 0.02 in controls, p<0.05). There were no severe developmental abnormaliites or visceral anomalies. No skeletal anomalies were seen although bone length and ossification centers were decreased at 0.1 mg/m3 and higher.

Effect levels (fetuses)

1

Dose descriptor:

LOAEC

Effect level:

0.1 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

LOAEC

Effect level:

0.1 mg/m³ air (nominal)

Based on:

labile/free

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

	Nominal inhalation dose (mg/m3)
Parameter tested	0	0.03	0.1	0.3	1
Embryotoxicity (%)	7	8	19*	22*	36*
Fetal weight, GD 21 (g, Fig 3)	3.45	3.35	3.03	3.15	2.95*
Ear unfolding (PND)	2.35	2.59	2.26	2.72*	2.63
Lower incisor eruption (PND)	8.27	8.32	8.36	8.99*	8.73
Upper incisor eruption (PND)	11.3	11.47	11.75	11.63	11.59
Eye opening (PND)	17.06	16.99	17.57	16.56	17.57
Pup weight, PND 1 (text)	4.29				5.27*
Dead pups, PND 5 (%, text)	17.75				76*
Dead pups, PND 5-21 (%, text)	4.18	no different than control			40*
Grooming behavior, 30 days, males		#	#	#	#
Grooming behavior, 30 days, females				#
Grooming and defecation, 60 days, males
Grooming and defecation, 60 days, females		#		#	#
Pain threshold, 60 days, male	2.43				2.87*
Pain threshold, 60 days, female	1.98				2.63*
Decrease in relative testis weight					#
Increase relative spleen weight, female			#		#
Increase relative adrenal weight, female			#	#
Increase relative ovarian weight			#
	* significant difference from control, p<0.05 or 0.01
	# significant difference from control as stated in the text or a figure, no number available.

Applicant's summary and conclusion

Conclusions:

Based on the expected difference between the nominal inhalation dose and the unknown real dose that combines inhalation and oral exposure from grooming, the conclusions are qualitative despite NOELs and LOELs stated by the study. Although the dose is unknown, embryotoxicity and fetotoxicity that persists into early postnatal life are demonstrated at doses that are not apparently maternally toxic. The parameters affected included pre-implantation and post-implantation loss, skeletal abnormalities, and greater postnatal mortality.

Executive summary:

25 female Wistar rats were exposed to 0, 0.03. 0.1, 0.3, and 1.0 mg/m3 tribromophenol from gestation day 1 to gestation day 21 continuously (24 hours per day, 7 days per week). Based on this continuous exposure, the doses stated are nominal and not likely to be the true doses because grooming would add an unknown oral exposure, and the group housing would allow some animals to block inhalation of compound by placing the nose into another animal's fur, thereby filtering the exposure to an unknown degree. On gestation day 21, the dams were observed in an open field to determine movement and orientation parameters, then 15 of the dams were sacrificed and examined for pain threshold, ovarian and uterine markers of embryotoxicity, immune system parameters, body weight, rectal temperature, levels of lipid peroxidation, nitrogen, phenols, hormones, and specific enzymes. The fetuses were examined for gross, skeletal, and visceral abnormalities. The ten remaining dams were allowed to litter naturally, and the days at which developmental markers were reached was recorded. The pups were tested in the same open field apparatus, and for pain threshold, at 30 and 60 days of age, then sacrificed and relative weights determined for specific organs.

The maternal effects statistically different from controls were seen at the highest exposure level - a decrease in orientation as measured by decreased rate of poking the nose into holes in the open field apparatus, and increase in level of alkaline phosphatase in blood, amino nitrogen in urine, and increased level of blood progesterone. Embryotoxic effects were seen as an increase in both preimplantation and postimplantation embryo loss at the nominal level of 0.1 mg/m3. Fetuses examined at gestation day 21 showed decreased weight and length in a dose-responsive manner starting at 0.1 mg/m3, and placental weight showed a downward trend with increasing nominal dose. No severe developmental malformations in the body or head were seen, and no visceral anomalies. Although no skeletal anomalies were seen, there was a trend toward decrease in length of long bones, number of metacarpal and metatarsal bones, and a decrease in the number of ossification centers with increasing dose. In postnatal pups at the highest dose, the mortality rate was increased with treatment - 75% vs 18% in controls by PND 5, and between PND 5 and PND 21 the death rate was 40% vs 4.5%, respectively. When tested in the open field, grooming behavior was decreased at both time points, and pain threshold was significantly increased at PND 60 in both males and females. Females had significant increases in the relative weights of spleen and adrenals, but there was no dose response.

Although this study states LOELs and NOELs for maternal and developmental toxicity, the uncertainty regarding the actual dose by combined inhalation and ingestion from grooming leaves these results qualitative rather than quantitative. Of greatest relevance is the finding that embryotoxicity and fetotoxicity are seen at levels that are apparently not maternally toxic, and therefore maternal toxicity markers are not likely to be protective of the fetus.