2-methylpyridine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421: Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pyridine

EC Number:

203-809-9

EC Name:

Pyridine

Cas Number:

110-86-1

Molecular formula:

C5H5N

IUPAC Name:

pyridine

Test material form:

other: liquid

Details on test material:

Pyridine, Batch 0000076722 , one generation
- Name of test material (as cited in study report): Pyridine
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state:
- Analytical purity: 99.98% purity
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.:0000076722
- Expiration date of the lot/batch: 23 February 2009
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material:
- Other:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories UK Limited, Margate, Kent, UK
- Age at study initiation: ca 6 weeks of age
- Weight at study initiation: 200-225 g for males and 150-170 g for females
- Fasting period before study:
- Housing: The animals were initially housed 2 per cage, in polypropylene cages, measuring ca 42 x 27 x 20 cm, with solid bottoms and stainless steel mesh tops. A stainless steel food hopper and a polypropylene or polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately.
A few days prior to pairing for mating, males were transferred to individual polypropylene grid-bottomed cages (ca 59 x 38.5 x 20 cm) with stainless steel mesh tops. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. During mating, each female was transferred to the cage of an appropriate co-group male and remained there until mating was detected or seven nights had elapsed. Mated females were transferred to individual ca 42 x 27 x 20 cm solid bottomed cages. White tissue paper was provided as nesting material from Day 20 of gestation. Females with litters remained in this type of caging until termination. Males remained singly housed until termination.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) SQC supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used in this study is retained in the study archive.
The food was not considered to contain any additional substances in sufficient concentration to influence the outcome of the study.
- Water (e.g. ad libitum): The animals had access to domestic, mains quality water ad libitum. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.
- Acclimation period: The animals were acclimatised in the Charles River Laboratories animal room for 13 days prior to commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C-23°C
- Humidity (%):43%-68%
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES:
MALES
From: 6 weeks of age To: post 4 weeks of treatment
FEMALES
From: 6 weeks of age To: day 5 or 6 of lactation (along with pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Doses were administered once daily at approximately the same time each day by oral gavage at a dose volume of 5 mL per kg body weight, using a plastic gavage and a plastic syringe. The volume to be administered to each animal was determined on each day by the weight of the animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until parturition was complete, the dose volume of the females was determined by the weight of the animal on Day 16 of gestation.
Group Number
Treatment (mg/kg/day)
Animal Numbers
Males
Females
1
Control 0
1-10
41-50
2
Low dose 10
11-20
51-60
3
Intermediate dose 25
21-30
61-70
4
High dose 50
31-40
71-80
Page 18 Study No. 494646
The males were dosed once daily for 4 weeks overall, commencing 2 weeks prior to mating.
The females were dosed once daily from 2 weeks prior to mating then continued until at least Day 4 of lactation; the females were sacrificed with their litters between Days 5 and Day 6 of lactation.
Dosing for males and females continued until the day prior to termination.
On Day 21 of gestation, Animal 63 (Group 3) did not receive the full dose amount due to reflux. This slight departure from the dosing regimen was not considered to have affected the integrity of the study.

Duration of treatment / exposure:

Male rats from 14 days prior to mating, throughout the 7 day mating period, and 7 days post-mating.
Female rats from 14 days prior to mating throughout the 7 day mating period, during pregnancy and up until at least day 4 of lactation.

Frequency of treatment:

Once daily

Duration of test:

Males: 4 weeks; Females: 14 days prior to mating until at least day 4 of lactation

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The females were allowed to litter normally. The day of birth of the litter was designated Day 0 of lactation. The duration of gestation in days was calculated and evaluated.
At termination, a gross examination was undertaken in both sexes. In males, liver, testis and epididymis were weighed and examined histologically. In females, liver and ovaries were weighed and examined histologically.

Examinations

Maternal examinations:

The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Data recorded after Day 4 of lactation have not been reported, but details are retained in the study data.
Any deficiencies in maternal care were recorded. Points looked for were inadequate construction and cleaning of the nest, pups left scattered and cold, physical harm of pups, or apparently inadequate lactation or feeding.

Ovaries and uterine content:

Ovaries and uterine contents were examined for the number of corpora lutea and implants.

Fetal examinations:

The pups were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. The pups were discarded following evaluation.

Statistics:

Body weight and food consumption of males, and of females prior to pairing were subjected to analysis of variance or the Kruskal-Wallis non-parametric analysis. Pregnancy performance data (number of corpora lutea, implants, pups born and live pups during lactation) were subjected to the Kruskal-Wallis analysis. Organ weight data were analysed by analysis of variance and analysis of covariance using the terminal body weight as the single covariate. All statistical tests were 2-sided and performed at the 5% significance level using validated in-house software. Pairwise comparisons were only performed against the Control group.

Indices:

For each group:
Fertility Index (male) = Number siring a litter/ Number paired
Fertility Index (female) = Number pregnant/ Number paired
Gestation Index = Number bearing live pups /Number pregnant

For each litter and group:
Birth Index = Total number of pups born (live and dead)/ Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation /Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/ Number live on Day 0

Historical control data:

Available and utilized for the appropriate comparisons.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Increased liver weight at all doses

Details on maternal toxic effects:
Slight intergroup differences in the body weight gain of the males were too small to be considered treatment-related. In the females treated at 50 mg/kg/day, there was a very slight reduction in group mean body weight during Week 1 of treatment but gains were similar to the control group for Week 2 of treatment (none of the differences were statistically significant). There was also a slight reduction in group mean body weight gain during mid-late gestation for the females treated at 50 mg/kg/day. Group mean body weight gains during lactation for the females in the 50 mg/kg/day group were similar to the control group. There were no effects on weight gain considered to be treatment-related for the females treated at 10 and 25 mg/kg/day.
For females treated at 50 mg/kg/day, group mean food consumption over lactation days 0-4 was slightly reduced as compared to controls. There were no treatment-related changes in food consumption for females treated at 10 and 25 mg/kg/day or for males treated at any dose level.
There were no treatment-related effects on mating performance, fertility or duration of gestation. The number of corpora lutea graviditatis and implants were lower for the females treated at 50 mg/kg/day as compared to the control females. In addition, the remaining mean litter sizes of dams with live pups, for the 50 mg/kg/day group, were low throughout lactation days 0-4 as compared to the controls. The reductions in the corpora lutea graviditatis, implants, pups born and live pups on lactation day 0 were all within the Historical Control background range and none of these endpoints achieved statistical significance. The reductions in the number of live pups on lactation days 1 and 4 were lower than the Historical Control background range with the reduction on lactation day 4 achieving statistical significance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Decreased mean numbers of live pups per litter on days 1 and 4 of lactation for the 50 mg/kg/day dose group.

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on litter size or survival for the 10 or 25 mg/kg/day groups.
At 50 mg/kg/day, mean pup weights were similar to the Controls; lower litter weights at this dose may have reflected the smaller litter sizes. There were no treatment-related effects on litter and pup weights for the groups treated at 10 or 25 mg/kg/day. There were no gross abnormalities amongst the pups and no treatment-related clinical observations.
There was a statistically significant dose-related increase in liver weights for males in all treatment groups; there was no treatment-related effect on the weights of the epididymides or the testes. In the females, there was also a statistically significant increase in liver weights in all treated groups. There were no treatment-related histological findings observed in the ovaries from the females or the testes and epididymides of the males.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of the dosing formulations was undertaken with regard to concentration and homogeneity. Formulations were initially scheduled to be analysed on two occasions during the study treatment period; from formulations prepared for use on 02 April 2007 (prepared 30 March 2007) and 16 April 2007 (prepared 13 April 2007). Results from the second analysis occasion (prepared 13 April 2007) were outside the acceptance criteria of ± 10%. Due to these results, further analysis occasions were considered appropriate. Although the results of analysis from formulations prepared for use on the second week of treatment were found to be low (ca -20% of nominal), all future formulations were found to be accurately prepared and the integrity of the study was not considered to have been affected by the low results from this week of treatment.

Under the conditions of this study, a parental No Observed Effect Level (NOEL) was not established due to the increased liver weights observed in all treatment groups. The NOEL for reproductive parameters was considered to be 25 mg/kg/day based on decreased mean numbers of live pups per litter on days 1 and 4 of lactation for the 50 mg/kg/day dose group.

Applicant's summary and conclusion

Conclusions:

A developmental toxicity study (OECD 421) was undertaken on pyridine, a category member with 2-, 3-, and 4-methylpyridine. Doses of 0, 12, 25 and 50 mg/kg bw/d were administered by oral gavage to male rats at doses for 2 weeks prior to mating, and to females for 2 weeks prior to mating and throughout the gestation and lactation periods. There were no adverse reproductive findings attributable to pyridine. Under the conditions of this study, a parental No Observed Effect Level (NOAEL) was not established due to the increased liver weights observed in all treatment groups. The NOEL for reproductive parameters was considered to be 25 mg/kg/day based on decreased mean numbers of live pups per litter on days 1 and 4 of lactation for the 50 mg/kg/day dose group. This study indicates that there is no adverse reproductive toxicity at doses several-fold higher than doses causing toxicity in the dams or adult males. Reading-across from pyridine to this substance is valid for the purposes of classification and risk assessment.