Pyridine, alkyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across from supporting substance Substance: 5-ethyl-2-methylpyridine (MEP) CAS no.: 104-90-5 EC no.: 203-250-0 Pyridine, alkyl derivatives is a substance of Unknown or Variable Composition, Complex Reaction Products, or Biological Materials (UVCB, subtype 2). The substance is not a discrete chemical, but characterized by a variety of structures. The substance 2-ethyl-4-methylpyridine (CAS no. 26091-11-2) and its isomer 5-ethyl-2-methylpyridine (MEP, CAS no. 104-90-5) were selected as representative model structures. The read-across is based on the hypothesis that source and target substance have similar properties, because they share a common functional group, common precursors and the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals. Please see the attached read-across justification for details.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Pharmaco LSR Ltd

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: 5-ethyl-2-methyl-pyridine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Male and female 10-11 weeks of age
- Weight at study initiation: (P) Males: 327 to 363 g; Females: 205 to 243 g;
- Fasting period before study: None
- Housing: Rats were housed in TRIS cages from Modular Systems and Developments Company Limited, Hereford, England, or RB3 and RB3 modified cages from North Kent Plastic Cages Limited, Erith, Kent, England. The cages consisted of stainless steel (TRIS) or high grade polypropylene (RB3 and RB3 modified) bodies with lids and floors of stainless steel grid (TRIS and RB3 modified) or polypropylene floors (RB3). Cages with grid floors were suspended in batteries over trays covered with absorbent paper; the latter was changed at appropriate intervals, except during mating when it was changed daily.
Autoclaved wood shavings were provided as bedding during the littering phase and were changed at least twice weekly. Cages were changed and cleaned as necessary.
The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
At various stages of the study the maximum number of rats per cage was:
Acclimatization: 5M/5F (TR18)
Pre-mating: 5M/5F (TR18)
Mating: 1M/1F (RB3 modified)
Males to termination: 5M/- (TR18)
Females to Day 14-18 (post coitum): -/1F (RB3 modified)
Littering (from Day 14-18 post coitum to Day 4 post partum): -/1F + litter (RB3)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C (range 18-25 °C)
- Humidity: 55 % (range 40 % - 70 %)
- Air changes: Approximately 15 room air changes per hour.
- Photoperiod: 12-hour dark: 12-hour light

IN-LIFE DATES:
- From: 18 October 1993
- To: 07 December 1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 35 % aqueous propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The four groups were as follows (concentration: number of animals):
Control: 10M/10F
30 mg/kg bw/day: 10M/10F
95 mg/kg bw/day: 10M/10F
300 mg/kg be/day: 10M/10F
The test item was prepared freshly each day in 35 % aqueous propylene glycol. Dosages were calculated based on the material as supplied. The animals were dosed daily by the oral route (gavage), at a volume-dosage of 10 mL/kg. Dosing took place for 15 days before pairing and throughout the study until termination on Day 4 post partum for females and for males, after successful littering of the females. Control animals received the vehicle at the same volume-dosage during the same treatment period. The volume administered daily to each animal was based on the animal's bodyweight on that day. Animals in the process of parturition were not dosed.

VEHICLE
- Concentration in vehicle: 3, 9.5, 30 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 5M/5F
- Length of cohabitation: 14 day mating period
- Proof of pregnancy: ejected copulation plugs and a vaginal smear will be prepared from each female and examined for the presence of spermatozoa. The day on which evidence of mating is found will be designated Day 0 of gestation. Once mating has occurred, the males and females will be separated and smearing discontinued.
- After successful mating each pregnant female was caged (how): 1F RB3 modified cage
- Any other deviations from standard protocol: None.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test item in 35 % aqueous propylene glycol was assessed analytically in a trial preparation at concentrations of 3 and 30 mg/mL prepared before the commencement of treatment. Duplicate unit dose samples were assayed to provide the initial, concentration value of the stability test. Further samples were assayed after 24 and 48 hours storage at ambient temperature to determine the stability of test item in the formulation. The concentrations of test substance were also determined in formulations prepared for one occasion of dosing during the first and last weeks of treatment.
Analysis was performed by a HPLC chromatography:
The total unit sample of known weight was transferred quantitatively into a volumetric flask with HPLC mobile phase. After ensuring complete dissolution, the solution was made to volume and an aliquot further diluted to a known volume with HPLC mobile phase to give a nominal concentration of test item in the final solution between 20 and
40 mg/mL. The concentration was then determined by high performance liquid chromatography.
Linearity: calibration was linear over the concentration range 10 to 50 mg/mL.
Limit of Assay: the procedure was adequately sensitive for the assay of formulations at concentrations between 3 and 30 mg/mL employed in the toxicity studies. The transfer and dilution of samples was quantitative and no recovery correction was applied to the analyses.
Five accurate standard solutions of test item in HPLC mobile phase containing approximately 10, 20, 30, 40 and 50 mg/mL were prepared and chromatographed. A graph of test item concentration (mg/mL) versus peak area measured by computing integrator was prepared to confirm linearity. The data were regressed linearly and the regression line plotted on the graph. A standard solution of intermediate concentration was injected at intervals throughout the run to monitor the chromatographic performance and update the response factor for computation.
The test item was shown to be stable in 35 % aqueous propylene glycol for at least six hours, the maximum period permitted between preparation and use for gavage dosing. The concentrations of the analyzed preparations from first and last weeks of treatment were satisfactory.

Duration of treatment / exposure:

Dosing took place for 15 days before pairing and throughout the study until termination on Day 4 post partum for females and for males, after successful littering of the females. Control animals received the vehicle at the same volume-dosage during the same treatment period.

Frequency of treatment:

Daily, 7 days per week.

Details on study schedule:

- Age at mating of the mated animals in the study: ca. 15 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Group 1: 0 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Group 2: 30 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Group 3: 95 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
Group 4: 300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10M/10F

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Not reported.
- Rationale for animal assignment (if not random): Random

Positive control:

Not applicable.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at weekly intervals throughout the study. Females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 post coitum and on Days 1 and 4 post partum. Group mean values and SD were calculated for all recorded bodyweights.

FOOD INTAKE: Yes
- Food intake of males and females was recorded weekly on a cage basis until the animals were paired for mating. After mating food intake was measured over the periods: Days 0-2, 3-6, 7-9, 10-13, 14-17 and 18-19 of gestation for females which was more frequent than specified in the protocol. Food intake was measured weekly for females that failed to mate and for males. No food consumption was recorded for males during Week 3 of study as the majority were paired for mating.Group mean food consumption of males and females before pairing were calculated on a weekly basis from cage mean values.

Oestrous cyclicity (parental animals):

Vaginal smears were taken for ten days before pairing from all females and examined to establish the regularity and duration of the estrous cycle.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight and epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Observations at Day 1 post partum
All offspring were observed at approximately 24 hours after birth (Day 1) and the following recorded for each litter:
a) Number of offspring (live and dead);
b) Individual bodyweights (live offspring only);
c) Sex ratio;
d) Observations on individual offspring.
- Signs
Litters were observed daily for evidence of ill-health or reaction to treatment.
- Bodyweight and sex ratio
The offspring were weighed individually and sexed on Days 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS: Yes
Daily records were maintained of mortality and consequent changes in litter size. Wherever possible, any offspring found dead were examined externally and internally.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Following successful littering by the females, the males were killed by inhaled carbon dioxide and examined externally and internally for macroscopic abnormalities.
- Maternal animals: On Day 4 post partum, females were killed by inhaled carbon dioxide and litters were killed by intraperitoneal injection of pentobarbitone sodium B.Vet.C (Sanofi Animal Health Limited, Watford, Hertfordshire, England). Females and litters were examined externally and internally for macroscopic abnormalities.
The female which failed to mate was killed by inhaled carbon dioxide 25 days after a possible day of mating. The subsequent vaginal cytology and necropsy confirmed that mating did not occur during the pairing period.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1: Organ weights and pathological procedures were prepared for microscopic examination and weighed, respectively.
- Preservation: samples of the tissues were preserved in 4 % neutral buffered formaldehyde. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate. Testes and epididymides were retained in Bouins fluid for 24 hours before preservation in 4% neutral buffered formaldehyde.
- Histology: the tissue samples or regions detailed, taken from all specified animals, were dehydrated, embedded in paraffin wax, sectioned at approximately five micrometre thickness and stained with haematoxylin and eosin.
- Microscopy: microscopic examination was performed on the tissue sections specified in the Pathology Procedures Table for all parental rats of control and high dose groups.

Postmortem examinations (offspring):

SACRIFICE
All animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1: Organ weights and pathological procedures were prepared for microscopic examination and weighed, respectively.

Statistics:

The significance of suggestive inter-group differences was tested using appropriate statistical tests. Differences with an associated probability of p < 0.05 were considered to be statistically significant. The following tests were used:
- One-way analysis of variance and/or t-test
Bodyweights
Bodyweight change
Food consumption
Litter size
- Mann-Whitney U-test
Post-implantation survival index
Live birth index
Viability index

Reproductive indices:

Oestrous cycles
For each group the percentage of females showing the following cycle types was calculated:
Regular: All observed cycles of four or five days.
Irregular: At least one cycle of two, three or six to ten days.
Acyclic: At least ten days without estrus (beginning before pairing).
- Pre-coital interval
For each group the percentage of females with pre-coital intervals in the following categories was calculated: 1-4, 5-9 and 10-14 days.
- Mating performance and fertility
For each group and sex the following were calculated:
Percentage mating = Animals mated/Animals paired x 100
Conception rate = Animals that achieved a pregnancy/Animals mated x 100
Fertility index = Animals that achieved a pregnancy/Animals paired x 100
Gestation index = Number of live litters born/Number pregnant x 100
- Gestation length
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with day of mating = Day 1 for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
- Group mean litter size and SD were calculated as: Number of live offspring/Number of live litters on day of examination.

Offspring viability indices:

- Survival indices
The following indices were calculated for each group:
Post-implantation survival index was calculated as: Number of offspring on Day 1 post partum/ Number of uterine implantation sites x 100
Live birth index was calculated as: Number of live offspring on Day 1 post partum/ Total number of offspring on Day 1 post partum x 100
Viability index was calculated as: Number of live offspring on day of examination/Number of live offspring on bay 1 post partum x 100
- Group mean offspring bodyweight and SD was calculated as: Total of individual litter mean offspring weights/Number of litters on day of examination
- Sex ratio: the ratio of male to female offspring was calculated for Day 1, and for live offspring on Days 1 and 4 post partum within each group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below for details.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below for details.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below for details.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The general condition of animals was similar in all groups. A number of signs were seen after dosing for all treated groups some of which were in evidence up to two hours after dosing on a few occasions. Increased salivation was seen from Week 1 after dosing in all Group 4 (300 mg/kg bw/day) animals and continued to be seen throughout the study. Other signs seen in this group included apparently reduced body temperature and abnormal respiration (fast, slow or noisy) which were seen during Weeks 2, 3 and 4. Underactivity was also seen in males during Week 3. Hunched or prostrate posture, ataxia, piloerection and partially closed eyes were seen infrequently.
Increased salivation after dosing was seen in the majority of Group 3 (95 mg/kg bw/day) animals and in a few animals in Group 2 (30 mg/kg bw/day) on a small number of occasions. No other signs were observed after dosing in these groups.
Two males in Group 4 (300 mg/kg bw/day) were killed in extremis, No. 34 in Week 3 and No. 39 in Week 4 exhibiting ataxia, partially closed eyes, increased salivation, abnormal respiration and apparently reduced body temperature. Male 34 also exhibited prostrate posture and underactivity. Macroscopic examinations revealed reduced/dehydrated gastro-intestinal contents, accentuated lobular liver patterns, apparently reduced testes, epididymides, prostate glands and seminal vesicles and, in each male, a small mass on one epididymis. Microscopic examination of both masses revealed the presence of spermatozoal granuloma. Both deaths were considered to be related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Group mean bodyweights of males in Group 4 (300 mg/kg bw/day) were consistently reduced compared to the Controls achieving statistical significance in Weeks 2 to 7. Consequently the overall bodyweight gain for this group during treatment was statistically significantly lower (p < 0.001) than the Controls. The overall bodyweight change of males in Groups 2 and 3 (30 and 95 mg/kg bw/day) was essentially similar to that of the Controls.
Bodyweight performance of females before pairing was similar to that of the Controls for all treated groups. During gestation, the overall bodyweight change seen for Groups 3 and 4 (95 and 300 mg/kg bw/day) was significantly lower (p < 0.05 and p < 0.01 respectively) than that of the Controls. Bodyweight performance of females in Group 2 (30 mg/kg bw/day) was similar to the Controls and showed no effect of treatment with test item. During lactation, the bodyweight change of females in Group 4 (300 mg/kg bw/day) was lower than the Controls. Lactation bodyweights of females in Groups 2 and 3 (30 and 95 mg/kg bw/day) were similar to the Controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Food consumption for males was essentially similar in all groups throughout the treatment period. Female food consumption before pairing was unaffected by treatment for all groups. During gestation, food consumption showed some inter-group variation but no treatment-related effects were apparent. A slight reduction in Group 4 (300 mg/kg bw/day) food consumption was seen during lactation consistent with the reduced litter
sizes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Some variation in the length of estrous cycles was seen in all treated groups but the majority of females showed regular four or five day cycles and no effect of treatment with test item was apparent.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Analyses of organ weights following termination of males revealed a significant reduction in the absolute weights of the epididymides and seminal vesicles (p < 0.05) in Group 4 (300 mg/kg bw/day) compared with the Controls. This was considered to be associated with the reduced bodyweight performance of these males and not as an effect of test item on these reproductive organs.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The majority of animals mated within four or five days of pairing at the first estrous and all females were pregnant. One pair of animals in Group 4 (300 mg/kg bw/day) failed to mate during the fourteen day pairing period, but mating performance and fertility were not considered to have been affected by treatment.
Females in Group 4 (300 mg/kg bw/day) indicated a trend towards slightly increased gestation lengths. The incidence of females with a 234 day gestation length exceeded the current background control subset range. The normal range, however, for gestation length is 22-23.5 days and therefore the significance of this finding in relation to treatment with test item was considered to be equivocal. Females in Groups 2 and 3 (30 and 95 mg/kg bw/day) showed gestation lengths comparable with those of the Controls. Parturition proceeded normally and there was no evidence of dystocia. The gestation index was maximal in all groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Bodyweight-relative liver weights for males and females and male kidney weights in Group 4 (300 mg/kg bw/day) were higher than those of Control animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic examinations of males and females from Controls and from animals in Group 4 (300 mg/kg bw/day) revealed no significant findings that could be attributed to treatment with test item.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological evaluations of epididymides, testes and ovaries from Controls and from animals in Group 4 (300 mg/kg bw/day) revealed no significant findings that could be attributed to treatment with test item.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: General reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

95 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: General toxicity

3

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: General toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see below for details.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below for details.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
The general condition of offspring was essentially unaffected by treatment with test item in Groups 2 and 3 (30 and 95 mg/kg bw/day). Offspring in Group 4 (300 mg/kg bw/day) exhibited signs of poor health as illustrated by poor weight gain and viability.

CLINICAL SIGNS (OFFSPRING)
No examined.

BODY WEIGHT (OFFSPRING)
Group mean bodyweights of males and females in Group 4 (300 mg/kg bw/day) were significantly lower than the Controls at Days 1 and 4 of age. The bodyweight gains to Day 4 of age were inferior when compared with the Controls, achieving statistical significance for females (p < 0.01). Offspring bodyweights in Groups 2 and 3 (30 and 95 mg/kg bw/day) were similar to those the Controls and no effect of treatment was apparent.

ORGAN WEIGHTS (OFFSPRING)
Examination of offspring at necropsy revealed no significant changes that could be attributed to treatment of the F1 generation with test item.

GROSS PATHOLOGY (OFFSPRING)
Examination of offspring at necropsy revealed no significant changes that could be attributed to treatment of the F1 generation with test item.

HISTOPATHOLOGY (OFFSPRING)
Examination of offspring at necropsy revealed no significant changes that could be attributed to treatment of the F1 generation with test item.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

95 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: General toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Mating performance and fertility of males and females in all treated groups were unaffected by administration of test item at dosages up to and including 300 mg/kg bw/day. Administration of test item to male rats at a level of 300 mg/kg bw/day was associated with low bodyweight gain throughout treatment which was considered to be an indicator of non-specific toxicity. Treatment of females at this level did not elicit a similar bodyweight effect until gestation. The poor bodyweight gains seen at this stage continued to termination on Day 4 of lactation. These effects were reflected in the growth of the fetus in utero, with a resultant poor bodyweight performance of the pups and a significant decrease in viability to Day 4 of lactation.
Two females at 300 mg/kg bw/day showed low numbers of implantations and consequently reduced litter sizes. This resulted in a slightly low group mean value when compared with the Controls. In view of the numbers of implantations seen in the remaining females at 300 mg/kg bw/day, it was considered unlikely that administration of test item affected numbers of implantations and resultant litter sizes.
All animals receiving 95 or 300 mg/kg bw/day exhibited episodes of increased salivation after dosing. A number of isolated occurrences were also seen in animals receiving 30 mg/kg bw/day. Increased salivation is often seen with orally administrated materials and is commonly associated with an unpleasant taste of the test formulations.
It was concluded that administration of test item at levels up to 300 mg/kg bw/day was without effect on the general reproductive performance of the animals. The no-effect level for general toxicity was considered to be 95 mg/kg bw/day for males and 30 mg/kg bw/day for females. For offspring parameters, the no-effect level was considered to be 95 mg/kg bw/day.
At 300 mg/kg bw/day treatment was associated with apparently reduced body temperature, abnormal respiration and a low incidence of other signs including prostrate/hunched posture, tremor, piloerection and eyes partially closed, resulting in two males being killed in extremis. In addition, inferior bodyweight performance was associated with treatment of males and of females during gestation and lactation to termination on Day 4. Associated with the low weight gains of the females, the birth weights and subsequent bodyweight gains and viability of offspring were poor.
At 95 mg/kg bw/day poor bodyweight gains were seen during gestation for females.
Bodyweight gains of females receiving 30 mg/kg bw/day and of males receiving 30 and 95 mg/kg bw/day, were essentially unaffected by treatment.

Executive summary:

A study according to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified.

The influence of test item upon reproductive function and fertility was assessed in sexually mature male and female rats of the CD strain. Test item was administered by gavage at dosages of 30, 95 or 300 mg/kg bw/day to groups of ten male and ten female rats for 15 days before pairing. Treatment was continued throughout mating, gestation and lactation prior to termination on Day 4 post partum. Control animals received the vehicle, 35 % aqueous propylene glycol, throughout the same period.

Animals in all treated groups showed increased salivation after dosing which was most marked at 95 and 300 mg/kg bw/day. Animals receiving 300 mg/kg bw/day also showed an apparent reduction in body temperature and abnormal respiration during Weeks 2 to 4 after dosing. In addition to these, a small number of other signs were seen infrequently at 300 mg/kg bw/day. Two males receiving 300 mg/kg bw/day were killed in extremis after dosing with signs including ataxia, partially closed eyes, prostrate posture and underactivity. Terminal investigations revealed reduced/dehydrated gastro-intestinal contents, accentuated lobular liver patterns, apparently reduced testes, epididymides, prostate glands and seminal vesicles and a small mass on one epididymidis. Examination of the masses revealed the presence of spermatozoal granuloma. The deaths were considered to be related to treatment with test item.

Bodyweights of males receiving 30 and 95 mg/kg bw/day were essentially similar to the Controls. Males receiving 300 mg/kg bw/day showed poor bodyweight gains throughout treatment. Female bodyweights before pairing were unaffected by treatment. During gestation bodyweight performance of females receiving 95 and 300 mg/kg bw/day were reduced. At 300 mg/kg bw/day the bodyweights during lactation were inferior to those of the Controls. Females receiving 30 mg/kg bw/day were unaffected by treatment with test item.

During lactation, females receiving 300 mg/kg bw/day showed slightly lower food consumption. Food consumption for males and females before pairing and during gestation was unaffected by treatment.

Estrous cycles were essentially unaffected by treatment. One pair of animals receiving 300 mg/kg bw/day failed to mate. All other animals mated at the first estrus and all females were pregnant.

Gestation length for all females was within the normal range of 22 - 23.5 days. One female receiving 30 mg/kg bw/day and three receiving 300 mg/kg bw/day were terminated as a result of a total litter loss. All females had inactive mammary tissue. Two of the females receiving 300 mg/kg bw/day showed liver changes a small spleen and pale areas in the kidneys. Numbers of implantations, survival and growth in utero, litter size, offspring viability indices, sex ratio and bodyweight at Day 1 of age and weight gain to Day 4 were unaffected by maternal treatment at 30 and 95 mg/kg bw/day. At 300 mg/kg bw/day reduced offspring bodyweights were apparent at Day 1 when compared with the Controls. Subsequent bodyweight gains to Day 4 were poor and were associated with a decrease in viability of these offspring. All other offspring parameters were unaffected by treatment at 300 mg/kg bw/day.

Necropsy of offspring revealed no changes that could be attributed to maternal treatment with test item.

No macroscopic or microscopic changes were observed at necropsy of the parental males and females that were considered to be related to treatment. Variations in absolute and bodyweight-relative organ weights were apparent in animals receiving 300 mg/kg bw/day and were considered to be associated with their reduced bodyweight performance.

It was concluded that administration of test item at levels up to 300 mg/kg bw/day for 15 days before pairing and throughout the study until termination, was without effect on the general reproductive performance of the animals. The no-effect level for general toxicity was considered to be 95 mg/kg bw/day for males and 30 mg/kg bw/day for females. For offspring parameters, the no effect level was considered to be 95 mg/kg bw/day.

At 300 mg/kg bw/day treatment was associated with apparently reduced body temperature, abnormal respiration and a low incidence of other signs including prostrate/hunched posture, tremor, piloerection and eyes partially closed, resulting in two males being killed in extremis. In addition, inferior bodyweight performance was associated with treatment of males and of females during gestation and lactation to termination on Day 4. Associated with the low weight gains of the females, the birth weights and subsequent bodyweight gains and viability of offspring were poor.

At 95 mg/kg bw/day poor bodyweight gains were seen during gestation for females.

Bodyweight gains of females receiving 30 mg/kg bw/day and of males receiving 30 and 95 mg/kg bw/day, were essentially unaffected by treatment.