Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported
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 Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Non-pregnant female rats ("serum donors") received 1000 mg/kg/day of test material via gavage for three consecutive days. Four hours after the last dose the rats were exsanguinated and their blood was centrifuged to obtain serum. Control rats were similarly gavaged with vehicle and bled. Rat conceptuses from a separate group of untreated, timed-pregnant rats were explanted on the afternoon of gestation day 9 and were cultured in the sera (100 %) from the control or test material serum donors. After approximately 42 hours in culture, embryos were evaluated for viability, growth and morphology.
GLP compliance:
no
Type of method:
other: ex vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
4-amino-3,6-dichloropyridine-2-carboxylic acid
EC Number:
604-721-7
Cas Number:
150114-71-9
Molecular formula:
C6H4Cl2N2O2
IUPAC Name:
4-amino-3,6-dichloropyridine-2-carboxylic acid
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: tan powder
Specific details on test material used for the study:
Purity: 95.4%

Test animals

Species:
rat
Strain:
other: Crl:CD
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: CD (Crl:CD (SD) IGS BR)
- Age at study initiation: 9 - 10 weeks (serum donors and embryo donors)
- Weight at study initiation: 200 - 250 g (non-pregnant serum doors and time-mated embryo donors)
- Housing: Animals were individually housed in stainless steel cages with wire-mesh floors that were suspended above catch pans and contained a feed crock and pressure activated nipple-type watering system.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % METHOCEL® A4M
Details on exposure:
DOSE PREPARATION
The test material was administered as a suspension in an aqueous vehicle of 0.5 % METHOCEL® A4M such that a dose volume of 4 mL/kg body weight yielded the targeted dose. Dose volumes were adjusted daily based on individual body weights of the serum donors.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Test material was administered via gavage to the non-pregnant serum donor rats for three consecutive days (test days 1 - 3).
Frequency of treatment:
Daily
Duration of test:
Approximately four hours after the last dose on test day 3, the rats were sacrificed via carbon dioxide inhalation and immediately exsanguinated
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7 females per group
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose level of test material was selected based on findings from an existing rat oral acute toxicity study. Furthermore, the dose level of 1000 mg/kg/day represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study).
- Rationale for animal assignment: Randomisation of the serum donor rats into dose groups was performed one day prior to the start of dosing (test day -1) using a computer generated procedure designed to increase the probability of uniform group mean weights and standard deviations at the start of exposure. The embryo donor rats were not dosed. Therefore, they were not randomised into groups nor were they individually identified.
Statistics:
Descriptive statistics were reported (i.e., mean ± SD) for all continuous data. Continuous data (embryo head length, crown-rump length, yolk sac diameter, somite counts and body weight gains from test day 1-3) were analysed by ANOVA. Flexion score was analysed by a non-parametric ANOVA. Percentage data (% viable, % with yolk sac circulation, % abnormal embryos, % embryos with generalised delay) were analysed via Fisher’s exact test.

Results and discussion

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no test substance related effects at the highesst dose level

Any other information on results incl. tables

Serum Donors

All serum donors appeared normal throughout the test period. Body weight gains in the test material treated rats were not significantly different from those of the control rats

Embryo Culture

There were no apparent differences between embryos cultured in test material serum vs. control serum for any parameter of embryo viability, growth or morphology. The only morphological abnormalities noted among the 14 control embryos were two embryos with an open neural tube. The other structures in these embryos exhibited generalised delay. Among the 15 embryos in the test material group, a single embryo exhibited generalised delay, but its pattern of development was otherwise normal.

Table 1: Results

Control

1000 mg/kg/day

Serum donors

No. of serum donors

7

7

No. deaths

0

0

Body weight change test day 1-3 (g)

-0.8 ± 6.6

-1.8 ± 8.4

Embryo viability

No. embryos cultures

14

15

% embryos with beating heart

100

100

% embryos with yolk sac circulation

100

100

Embryo growth

Yolk sac diameter (mm)

3.4 ± 0.4

3.6 ± 0.2

Crown-rump length (mm)

2.9 ± 0.3

3.0 ± 0.2

Head length (mm)

1.5 ± 0.1

1.5 ± 0.1

Somite number

19.9 ± 5.5

21.8 ± 3.4

Flexion score (0-3)

2.0 ± 1.0

2.2 ± 0.8

Morphology

% embryos with abnormalities

14.3 (2/14)

0 (0/15)

% embryos with generalised delay

14.3 (2/14)

6.7 (1/15)

Applicant's summary and conclusion

Conclusions:
Based on the findings, the test material was considered negative for teratogenic potential in this screening test.
Executive summary:

The test material was evaluated for preliminary teratogenicity screening in the ex vivo whole embryo culture test. Seven non-pregnant female rats ("serum donors") received 1000 mg/kg/day of test material via gavage for three consecutive days. Four hours after the last dose the rats were exsanguinated and their blood was centrifuged to obtain serum. Seven control rats were similarly gavaged with 0.5 % methylcellulose vehicle and bled. Rat conceptuses from a separate group of untreated, timed-pregnant rats were explanted on the afternoon of gestation day 9 and were cultured in the sera (100 %) from the control or test material serum donors. After approximately 42 hours in culture, embryos were evaluated for viability, growth and morphology.

All serum donors appeared normal throughout the test period. Body weight gains in the test material treated rats were not significantly different from those of the control rats. There were no apparent differences between embryos cultured in test material serum vs. control serum for any parameter of embryo viability, growth or morphology.

Based on these findings, the test material was considered negative for teratogenic potential in this screening test.