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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose and reproduction screening (≠ OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-10-04 to 1992-01-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD guideline 412 with additional information on male fertility. However not all endpoints on reproductive toxicity are covered and deviations to the OECD guidelines for reproductive toxicity (OECD 415 and 422) are applicable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 412 (1981)
Deviations:
yes
Remarks:
age of males was a little high (17w instead of 12-13w)
Principles of method if other than guideline:
Repeated-dose inhalation toxicity study combined with male fertility. Male fertility was examined by mating exposed males and controls with untreated young virgin pro-oestrus females two days after the exposure period. Mating was repeated 6-9 and 27-30 days after the last day of exposure for the recovery and control group.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-chloropropane-1,2-diol
EC Number:
202-492-4
EC Name:
3-chloropropane-1,2-diol
Cas Number:
96-24-2
Molecular formula:
C3H7ClO2
IUPAC Name:
3-chloropropane-1,2-diol
Details on test material:
- Name of test material (as cited in study report): α-chlorohydrin
- Source: Duphar B.V., Weesp, the Netherlands
- Physical state: Colourless liquid
- Analytical purity: > 99.5%
- Ratio of enantiomers: R-enantiomer:S-enantiomer = 50:50 (±1)
- Lot/batch No.: TCE89J10A:
- Storage condition of test material: Brown-glass bottles at 4-6 °C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation (i.e. at arrival): 17 weeks (males), 12 weeks (females exposed) and 10 weeks (females used for mating procedures)
- Weight at study initiation: ±565 g (males) and ± 313 g (females)
- Housing: before and after exposure individually housed in suspended stainless steel cages (45-32-18) fitted with wire-mesh floor and front
- Diet: Institute's stock diet, ad libitum
- Water: unfluoridated tap water, ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose/head only
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only units from ADG Developments Ltd, Codicote, Hitchin, Herts. SG4 8UB, United Kingdom
- Method of holding animals in test chamber: secured in plastic animal holders (Batelle)
- System of generating particulates/aerosols: High-concentration (18 ppm) obtained by passing 35 l air/min through rotation evaporater (kept at 40°C) filled with α-chlorohydrin. Low- and mid-concentrations generated by using non-rotating evaporators. The lower concentrations (I.e. 1 and 4 ppm) were generated by delivering controlled quantities of test material in water (respectively 1 g/l and 4 g/l) by a roller pump to a heated stainless stell capillary. Compressed air (20 and 30 l/min) was passed over this cappillary. Capillary and compressed air lines were surrounded by a heating coil, with temperature set at 70 °C to reach immediate evaporation
- Temperature in air chamber: 23,1 °C (1 ppm), 23,7 °C (4 ppm) and 22,8 °C (18 ppm)
- Humidity in air chamber: <1 % (initially ca. 20% in low- and mid-concentration)
- Air flow rate (l/min): 20 (low-), 30 (mid-) and 35 (high-concentration)
Details on mating procedure:
Two days after the exposure period all exposed and control males were placed overnight with untreated young adult virgin pro-oestrus females. The morning preceding the mating the oestrus cycles of the untreated females was determined by taking a vaginal smear. Smears were fixed and stained according to the method of Papanicalaou and microscopically exmined for pro-oestrus. From the females that were in pro-oestrus 40 animals were randomly selected and placed with the exposed males. Matings were on a one-male to one-female basis. The morning after mating vaginal smears were taken to determine whether mating had occured. On day 7 and 14 after exposure all exposed and control males were allowed to mate again
On days 6-9 and 27-30 days after the last day of exposure the recovery and control group was allowed to mate again. Matings were on a one-male to three-female basis and vaginal smears were taken each morning after pairing to confirm mating had occured. The mating procedure was continued until all 3 females were mated or 4 days had elapsed. For these last two matings females were not selected for pro-oestrus.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentration of α-chlorohydrin was measured 1-2 times per exposure day using a total carbon analyser (Ratfisch RS 55) with heating sampling line. Before the study the response of the flame ionization detector (FID) was calibrated by passing a measured amount of air and known volumes of solutions of test material to the carbon analyser. A calibration graph was obtained by plotting the concentration (ppm) against the outpout of the carbon analyser (mm). The calibration graph obtained was described by the following equation: Y= 0.40 + 0.18x. The correlation coefficient was 0.99857. The actual concentrations (and standard deviations) were: 1.3 (0.2), 4.8 (1.1) and 18.1 (1.2) ppm
Nominal concentration was determined by dividing the total amount of test material used per exposure unit by the total volume of air passed through each exposure unit. Volume of air passed through each exposure unit was checked once per day using a gasmeter. The nominal concentrations (and standard deviations) were 1.0 (0.3), 5.0 (1.2) and 31.5 (8.9) ppm

The large difference between nominal and actual concentration at the high-concentration level might be explained by saturation and resulting losses onto tubing and/or walls of the exposure unit.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Details on study schedule:
- Age at mating of the mated animals in the study: 20, 21 or 22 weeks (males) and 13, 14 or 15 weeks (females)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
18.1 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
4.8 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1.3 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5 for inhalation exposure plus 5 in the male recovery group; for mating 30 untreated females were used
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the maximum saturated level as indicated by the sponsor
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 30 days
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Minimum once per day
- Cage side observations checked: All signs of ill, health, reaction to treatment, and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: YES
-Time schedule: Weekly

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Only before the first mating
Sperm parameters (parental animals):
not examined
Litter observations:
not examined
Postmortem examinations (parental animals):
HISTOPATHOLOGY / ORGAN WEIGHTS
- testis, epididymis in males
- macroscopic examination of major maternal organs of the abdomen and thoracic cavity in females.

HAEMATOLOGY
- Time schedule for collection of blood: At autopsy on day 16 (3 days after last exposure)
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: No data
- How many animals: 5/sex/concentration
- Parameters checked include haemoglobin concentration, packed cell volume, red blood cell count, total white blood cell count, differential white blood cell count, prothrombine time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean platelet volume (MPV), plateletdistribution width (PDW), red blood cell distribution width (RDW-SD).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: At autopsy on day 16 (3 days after last exposure)
- Animals fasted: No data
- How many animals 5/sex/concentration:
- Parameters checked include alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (γGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin total, sodium (Na), potassium (K), chloride (Cl), inorganic phosphate.
Postmortem examinations (offspring):
not examined
Statistics:
Food intake was analyzed by one way analysis of variance followed by Dunnett's multiple comparison test. Body weights, organ weights, haematology and clinical chemistry was analyzed by one way analysis of variance followed by Dunnett's multiple comparison test or t-test, f the number of observations was too small to apply Dunnett's test. The number of implantations and corpera lutea, pre- and post implantation loss were analyzed by the Kruskall-Wallis test followed by the Mann-Whitney U-test. Fertility index, mortality data and histopathological changes were analyzed by the Fisher exact probability test.

Level of significance considered: p < 0.05
Reproductive indices:
Number of live implantations, number of dead implantations (resorptions), number of corpora lutea
Offspring viability indices:
not examined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Shortly after exposure all animals including the controls appeared ungroomed with red crusts around the eyes. This was due to the stay in the restraining tubes and the low humidity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the exposure period animals of all groups including controls lost weight or showed a decreased body weight gain. On day 14 no effect on growth was observed in both males and females. In the first week of the study food consumption was significantly decreased in the high concentration group in both males and females. No other differences were observed during the remaining exposure period and recovery period

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number of matings 2 days after exposure in all groups, including the controls was much lower than normal historical control values. This effect was possibly due to stress by the exposure and handling. All mated females from control group and low dose group became pregnant. 4/6 mated females of mid dose group and 0/6 mated females of high dose group were pregnant at autopsy on day 13 of pregnancy. This effect was considered treatment related and was not observed at the second and third mating where most females were pregnant. No other effects on mating, pregnancy and mean number of live implantations, resorptions and corpora lutea were observed during each of the three matings.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
4 ppm
Sex:
male
Basis for effect level:
other: The adverse effects on fertility were not accompanied by histopathological changes in the testes and epididymides. The reduced fertility was reversible within 9 days after last exposure

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for effects on male fertility in rats was 4 ppm. Reduced fertility was not accompanied by any histopathological changes in the testes and epididymides. Reduced fertility at 18 ppm was reversible within 9 days after the last exposure.

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