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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, fully adequate for assessment
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
the deviations were considered not to have influenced the outcome of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
butene-2
IUPAC Name:
butene-2
Details on test material:
- Name of test material (as cited in study report): butene-2
- Physical state: gas
- Analytical purity: cis & trans-2-butene ≤95%
- Molecular weight: 56.1
- Boiling point: 4°C
- Melting point: -139°C
- Vapour pressure: 2.5 bars at 20°C
- Explosive limits in air: 1.6 - 10%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, F.R.G
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: 278.8 - 321.6 g (mean 299.4g) for males and 191.5 - 219.0 g (mean 204.0 g) for females
- Fasting period before study: None
- Housing: 4 per sex/cage prior to exposure, and individually after mating, in suspended stainless steel cages.
- Diet: Stock diet ad libitum except for about 30 minutes prior to, and during exposure.
- Water: ad libitum except for about 30 minutes prior to, and during exposure.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Humidity: 37-80% (with occasional higher values after cleaning)
- Air changes: 10 per hr
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: From: 30 January 1992 To: 15 March 1992

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Modified H 1000 multitiered inhalation chambers manufactured by Hazleton Systems Inc., USA. Stainless steel, with glass doors, with a capacity of approximately 2.2 m3
- Method of holding animals in test chamber: Individually in wire mesh stainless steel cages
- Each chamber was fitted with a micromanometer which showed the negative pressure inside ( 1-4 mm water column).
- To generate the test atmospheres, the test material was passed from the cylinder via a pressure reducer, stainless steel tubing and two calibrated massflow controllers and rotameters to the inlet of the inhalation chambers, where they were diluted with filtered air from the air conditioning system to the desired concentrations. The atmospheres were directed downward to the location of the animals.
- At the bottom of the chamber the test atmosphere was exhausted.
- Control rats were exposed to filtered air only.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere samples were taken sequentially from each of the chambers (control chamber included). They were drawn through heated sampling lines and were passed via a controlled valves system (Kuax-Control, Kuhnke 61.000) to the total carbon analyser (Ratfisch RS 55, FRG). The response of the analyser was recorded by a Kipp analogue recorder.
- The concentration was determined approximately twice each hour in each test atmosphere. Samples were taken from the chambers at a location close to the cage units in which the animals were housed.
- During preliminary experiments, it was checked whether a uniform distribution was obtained by measuring the concentration at several locations.
- The response of the total carbon analyser was recorded in scale units and converted into concentration values (ppm) based on the response of the calibration mixtures.
- The nominal concentration was determined using the mean amount of test material used per hour divided by the mean hourly volume of air passed through the exposure chamber (control chamber excluded).
Details on mating procedure:
- M/F ratio per cage: 1:1 (from about 3.30 p.m. until 8.30 a.m)
- If no sperm was detected the female was caged again with the same male the next afternoon
- Length of cohabitation: until mating occurred or 1 week had elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Males and females, not showing evidence of copulation were housed individually after the mating period had elapsed and were exposed until necropsy at the end of the study.
- After successful mating each pregnant female was caged: Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean actual concentrations (± standard deviation) of butene-2 in the test atmospheres were 2476 (68) and 5009 (88) ppm, or 5.7 and 11.5 g/m3, for the low- and high-concentration respectively.
Duration of treatment / exposure:
Exposure: daily during the premating period (2 weeks), through mating (1 week) and gestation (up to and including day 19)
Study duration was 39-46 days for males and up to day 19 of gestation for females
Frequency of treatment:
6 hrs/day
Details on study schedule:
After an initial exposure period of 2 weeks, males and females were mated for 1 week. Females were allowed to litter and to rear their progeny until day 4 of lactation; after which the parent animals and the pups were killed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2500 or 5000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 2476 or 5009 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
5.7 and 11.5 g/m3
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed
Details on study design:
none
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As necessary, to appraise physical condition.

BODY WEIGHT: Yes
- Time schedule for examinations: Male rats were weighed weekly. Female rats were weighed weekly during the premating period, on days 0, 7, 14 and 21 of pregnancy and on day 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption was measured weekly (the second week during premating over a 6-day period) during the premating period in both males and females. Food consumption of parent females was also recorded weekly during pregnancy and through day 1-4 of lactation. Food consumption of parent males and not-mated females was recorded weekly after the mating period until scheduled sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy
- Anaesthetic used for blood collection: Yes; ether anaesthesia
- Animals fasted: No data
- How many animals: All F0 animals
- Parameters checked: haemoglobin, packed cell volume, red blood cell count, red blood cell distribution width (RDW-SD), reticulocyte count, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy
- Animals fasted: No data
- How many animals: All F0 animals
- Parameters checked: alanine amino transferase (ALAT)/glutamic-pyruvic transaminase (GPT), albumin, aspartate amino transferase (ASAT), bilirubin total, calcium (Ca), chloride (Cl), creatinine, y-glutamyl transferase (yGT), inorganic phosphate, potassium (K), sodium (Na), total protein, urea.
Oestrous cyclicity (parental animals):
not examined.
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
- The total litter size and numbers of each sex as well as number of stillbirths and grossly malformed pups were recorded on days 1 and 4 post partum.
- The litters were weighed on days 1 and 4

GROSS EXAMINATION OF DEAD PUPS:
- yes. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. Pups sacrificed on day 4 were also examined macroscopically.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- All male and female parent rats were killed by exsanguination from the abdominal aorta under ether anaesthesia. A complete gross examination was performed.
- Pregnancy was demonstrated on the basis of implantation sites observed at autopsy of the female rats.

ORGAN WEIGHTS: Yes
- The following organs were weighed: testes, epididymides, liver, kidneys, thymus, lungs with trachea and larynx.

HISTOPATHOLOGY: Yes
- The following organs were examined microscopically from control and 5000 ppm animals: testes, epididymides, liver, kidneys, thymus, lungs with trachea and larynx, ovaries, uterus, seminal vesicles, adrenals, brain, heart, spleen, nose and any abnormal tissues and organs.
Postmortem examinations (offspring):
SACRIFICE
- All offspring were killed at 4 days of age.

GROSS NECROPSY
- Macroscopic abnormalities were recorded.
Statistics:
Parental clinical findings, mating and litter data, and pathological changes were evaluated by Fisher's exact probability test; bodyweight and food consumption by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. Duration of gestation, litter size implantations and pre- and post-implantation loss were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test, and pup bodyweights by analysis of variance followed by Dunnett's multiple comparison tests.
Reproductive indices:
The following indices were calculated for each group: mating index, female fertility index, fecundity index and gestation index.
Offspring viability indices:
The following indices were calculated for each group: live birth index, viability index (days 1-4) and sex ratio.

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:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the males were comparable in all groups. Some slight differences in body weight change were observed. Fourteen days and 7 and 14 days after the start of exposure to 2500 and 5000 ppm respectively, female rats showed statistically significantly decreased mean body weights compared with controls. No effects on body weights of females were observed during gestation. On day 1 of lactation the mean body weight of the females of the 5000 ppm group was statistically significantly decreased. No effects on body weight change of the females was observed during the study.

FOOD CONSUMPTION
There was a statistically significantly decreased food intake of the females of the 5000 ppm group during the first week of the premating period only.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
5 000 other: ppm (11474 mg/m3, 11.5 mg/L) nominal.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on dams at the highest concentration tested, no effects on reproductive performance or fertility

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

BODY WEIGHT (OFFSPRING)
Mean body weight of the pups was slightly but not statistically significantly lower in the 2500 and 5000 ppm group, (possibly due to the higher number of pups in these groups).

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
5 000 other: ppm (11474 mg/m3, 11.5 mg/L) nominal.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on pups at the highest concentration tested, no effects on pup viability, body weight or development

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

In the original report a NOAEL for toxicity was set at 2,500 ppm based on the slight effects on body weights in males and females, and possibly food consumption in females. A re-analysis of the data by RIVM (The Netherlands) concluded that as these effects were not dose-related and not consistently present during the study, the NOAEL for butene-2 should be ≥5000 ppm.

 

The data in question is shown in Table 1.

 

  • The changes in the body weight gain of the males was not considered to be adverse. It was only observed in week 1 and in week 4, when only the low dose was decreased, not showing a dose-response relationship. The change of 12 to 20 g, compared with the body weight of about 300 g is small. Furthermore, this effect was not observed in week 2 and 3, and no significant effects were shown on the body weight of the males.
  • In females, the effect on food consumption was not accompanied by an effect on body weight.
  • The effect on body weights of females was also not consistent through the whole study and was small (at the most about 5%).

 

Table 1. Treatment-related effects on body weight, body weight gain and food consumption

 

 

0

2500

ppm

5000

ppm

MALES

 

 

 

 

Week 1

Body weight

317.9± 4.36

313.9± 2.91

314.4± 3.63

Weeks 0-1

Body weight change

21.78 ±1.56

 

12.66±1.20

(58.1%)**

13.48± 1.54

(61.9%)**

Week 5

Body weight

369.6± 6.70

361.8± 4.14

367.0± 4.59

Weeks 4-5

Body weight change

10.87±1.38

5.84±1.74

(53.7%)*

11.60±1.20

(106.7%)

FEMALES

 

 

 

 

Week 0

Body weight

205.33± 2.10

203.60± 1.55

203.15± 1.91

Week 1

Body weight

214.25± 2.23

210.83± 1.55

(98.4%)

207.79± 1.13

(97.0%)*

Weeks 0-1

Body weight change

8.93± 1.21

7.23± 1.43

4.64± 1.49

Week 2

Body weight

220.28± 2.13

212.78± 2.05

(96.6%)*

213.19± 1.42

(96.8%)*

Weeks 1-2

Body weight change

6.0± 1.46

1.94± 1.79

5.40± 1.00

Body weight

Lactation Day 1

243.13±2.00

238.42±3.44

(98.1%)

230.61±2.67

(94.9%)*

Weeks 0-1

Food consumption

70.42±1.40

68.76±1.52

(97.6%)

65.28±0.96

(92.7%)*

Anova + Dunnet test; *= p, 0.05, **=p< 0.001

Applicant's summary and conclusion

Conclusions:
No effects on reproduction or pup development were seen when male and female rats were exposed to 2-butene at concentrations of 2500 or 5000 ppm (approximately 5737 or 5737mg/m3) for two weeks prior to breeding, during breeding and until day 19 of gestation.
Executive summary:

Male and female rats were exposed to 2-butene at target concentrations of 2500 or 5000 ppm (approximately 5750 or 11500 mg/m3) for two weeks prior to breeding, during breeding and until day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until post-natal day 4. Some decreases in body weight were observed in females at 2500 or 5000 ppm during the mating period but no other treatment-related changes were observed. There was no evidence of significant systemic toxicity in the parents. There were no effects on mating behaviour, fertility and gestation indices, the number of implantation sites and corpora lutea per dam, numbers of pups delivered, viability of pups at and after birth and the pup sex ratio when compared to the control group. Microscopic evaluation of gonadal function in parental males revealed no difference between treated and control groups. The NOAEC for reproductive toxicity was 2500 ppm (5700 mg/m3) based on the decreased body weights. There were no treatment-related effects on the development of pups. There were no effects on body weight gain or observed during macroscopic examination of pups at post mortem. Based on these data, the NOAEC for developmental toxicity was 5000 ppm (11,500 mg/m3), the highest concentration tested.

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