Hydrocarbons, C13-23

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Source of data is from peer reviewed literature. Acceptable well-documented study report which meets basic scientific principles. GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

A three generational study examining reproductive toxicity.

Principles of method if other than guideline:

A three generational study examining reproductive toxicity.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C9 aromatics

IUPAC Name:

C9 aromatics

Details on test material:

- Name of test material (as cited in study report): Test material was specifically prepared to meet the composition requirements for High Flash Aromatic Naptha, Type 1
- Composition of test material, percentage of components: at least 22% ethyltoluene isomers, 15% trimethylbenzene isomers; 75% total ethylbenzenes and trimethylbenzenes as according to ASTM method D-3734 specifications for High-Flash Aromatic Naptha, Type 1

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation:(P) 6 x wks; (F1) 5-7 x wks; (F2) 22 days
- Housing: Inidividually housed in wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2-3 weeks

ENVIRONMENTAL CONDITIONS
Animal husbandry followed standards by the US Department of Health, Education, and Welfare (1985)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Exposure apparatus: 16 m glass and steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: Air was provided by a seperate HVAC system.
- Method of conditioning air: Air was filtered for particulates and temperature and humidity controlled.
- System of generating particulates/aerosols: Test atmosphere was generated by heating nitrogen to 200°C by passing it through a 1 l stainless steel cylinder with a 1500 W band heater. The nitrogen then passed through a glass column 7.6 cm diameter and 30 cm long packed with glass beads. Test material was delivered by a metering pump into Teflon tubing, to the bottom of the column. The liquid test substance vaporized as it went up the column with the nitrogen. The vapor then went into the test chambers where dilution with the chamber ventilation air produced the desired concentrations.
- Temperature, humidity, pressure in air chamber: Air flow rate, temperature and relative humidity were monitored every half-hour during exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: Measurements made hourly using gas-phase IR.
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm-positive vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of the test material in the test chambers was determined by GC analysis.

Frequency of treatment:

Both males and females were exposed 6 hours per day 5 days/ week for 10 weeks before mating. After mating, females were exposed 6 hours/day, 7 days/week from Gestation Day (GD) 0 to GD 20. Dams were then removed to nesting boxes to deliver. Dams were again exposed from postnatal day (LD) 5 until weaning on LD 21.

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation one week after weaning.
- Age at mating of the mated animals in the study: 15-17 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 500, 1500 ppm
Basis:
nominal conc.
0, 500, 2500, 7500 mg/m3

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 103, 495, 1480 ppm
Basis:
analytical conc.
0, 515, 2475, 7400 mg/m3

No. of animals per sex per dose:

30, except for F2 generation in which 40 animals/sex/dose were selected. In the 1500 ppm F2 group all surviving animals were mated.

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for viability and overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until confirmation of mating; females weighed on GD 0, 7, 14, 21, and LD 0, 7, 14, 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly except during mating, gestation, and lactation
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

Litter observations:

STANDARDISATION OF LITTERS
Litters were culled by random selection to 8 pups on LD4.


PARAMETERS EXAMINED
The following parameters were examined in F1/F2/F3 offspring:
Number of pups, stillbirths, live births, presence of gross anomalies was examined as soon as possible after delivery. Pups weighed individually on LD 0, 4, 7, 14. On LD21 all pups were counted, sexed, and weighed.


GROSS EXAMINATION OF DEAD PUPS:
Yes, culled pups and any pups that died were necropsied for gross abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were necropsied at end of mating period.
- Maternal animals: All surviving animals were necropsied following weaning.

HISTOPATHOLOGY
In control and high dose group the epididymis, lung, ovary, pituitary, prostate, seminal vesicle, testis, uterus, vagina, lymph nodes were examined. Masses and gross lesions were examined from all groups.

ORGAN WEIGHTS
Epididymis, lung, ovary, testis, prostate/seminal vesicle, uterus/vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1/F2 offspring not selected as parental animals were sacrificed at 8 days of age. F3 offspring were sacrificed at LD21.

GROSS NECROPSY
Culled pups and any pups that died were necropsied for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
F1/F2 offspring were examined the same as parental animals.

Statistics:

Fertility indices and female/male ratio was analysed using Chi-square test criterion with Yate's correction. Proportions of litters with malformations were compared using Fisher's exact probability test. Proportions of resorbed fetuses, implantation losses, and pup survival were compared using Mann-Whitney U test. Mean number of liveborn pups/litter and pup weight were compared using analysis of variance and appropriate t-tests. Other parameters were compared using analysis of variance, appropriate t-test, and Dunnett's multiple comparison tables.

Reproductive indices:

female mating index: number pregnant females/number of females mated
female conception index: number females delivering live litter/number pregnant females
female gestational index: number of females delivering live litter/number females delivering a litter
male fertility index: number of fertile males/number of males mated
Cohabitation time: average number male/female cohabitation days litter size at birth

Offspring viability indices:

Gestational survival index: number pups live born/number of pups born
Postnatal survival index (4-day): number pups alive at LD4/number of liveborn pups
Postnatal survival index (21-day): number pups alive at LD21/number of liveborn pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

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)

F0
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All males survived with minimal signs of toxicity. 7 females in the 1500 ppm group died.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain of P males and females was significantly reduced in the 500 ppm and 1500 ppm groups. Food consumption was reduced in the 1500 ppm group in the first week.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant differences in number of mated females, number of females delivering a litter, number of females delivering a live litter, or male fertility. The mean number of live births per litter were not significantly reduced. Length of time for mating was not significantly different from controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no significant pathological findings in the reproductive organs in any group of animals in any generation in any dose group.

F1
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 1500 ppm group ataxia and reduced motor activity was observed. 6 females in this group also died.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of 1500 ppm group, and males in the 500 ppm group were significantly less than control groups. Food consumption was normal.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Length of time for mating was increased, but not significantly. Male fertility was reduced, however, as this effect was not seen in the first and third generations, this is not considered to be exposure related.

F2
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Most of the animals (36/40 males and 34/40 females) in the high exposure group (1500 ppm) died within the first week.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain of high exposure males and females were severely below control (up to 40%). Body weights in the 500 ppm and 100 ppm exposure groups were mildly depressed (10%). Food consumption was similar for all groups.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Effect level:

>= 1 500 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 7500 mg/m3

2

Dose descriptor:

NOAEC

Effect level:

>= 1 500 ppm

Sex:

male/female

Basis for effect level:

other: 7500 mg/m3; If maternal exposure ceased before gestation day 20, there were no negative effects on the offspring.

Remarks on result:

other: Generation: offspring of all generations (migrated information)

3

Dose descriptor:

LOAEC

Effect level:

1 500 ppm

Sex:

male/female

Basis for effect level:

other: 7500 mg/m3; Reduced body weight in offspring was noted if maternal exposure was continued until delivery.

Remarks on result:

other: Generation: offspring of all generations (migrated information)

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:

no effects observed

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)

F1
VIABILITY (OFFSPRING)
No significant differences through the lactation period.

BODY WEIGHT (OFFSPRING)
No significant difference in body weights or birth weight, except for pups from the 1500 ppm group at LD7 through weaning.

F2
VIABILITY (OFFSPRING)
Mean birth weight in the 1500 ppm group was reduced, but not significantly. The mean number of live offspring/litter, and number live-born/number delivered was significantly reduced in this group. This was most likely due to litters from dams which had not been confirmed to have mated. These unconfirmed dams were exposed until delivery, whereas the other dams were not exposed after GD20. The birth weights from these litters were not significantly reduced.

BODY WEIGHT (OFFSPRING)
Mean body weights in the 1500 ppm group after LD7 were significantly reduced.

F3
VIABILITY (OFFSPRING)
There were no apparent effects on mating, fertility, mean number of live births, and survival through lactation period.

BODY WEIGHT (OFFSPRING)
Mean weight of the offspring in the high exposure group was significantly reduced at birth. This may have been the result of the small sizes of the dams. If maternal exposure was ceased, the body weight gain became normal, but if reinitiated, the body weights were again reduced.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Significant Effects of Exposure to High Flash Aromatic Naptha (SD)

Male Fertility Index	F0	F1	F2
0	86.7 (30)	89.7 (30)	93.3 (30)
100	96.7 (30)	86.7 (30)	83.3 (30)
500	83.3 (30)	93.3 (30)	80.0 (30)
1500	84.6 (26)	64.3 (28)	100 (4)
Litter Size at Birth	F0	F1	F2
0	12.1 ± 3.4	12.4 ± 2.0	12.6 ± 2.7
100	12.9 ± 1.5	11.1 ± 2.9	11.8 ± 2.3
500	12.2 ± 3.1	11.7 ± 3.0	11.4 ± 2.1
1500	11.3 ± 3.0	8.7 ± 4.3	12.2 ± 1.3
Gestational Survival Index	F1	F2	F3
0	95.9 (366)	97.4 (383)	97.7 (361)
100	97.9 (382)	95.4 (280)	98.2 (335)
500	94.9 (333)	91.6 (371)	98.5 (325)
1500	92.8 (279)	85.1 (215)	100 (73)

 

Gestational Survival Index Among Rats in the F2 Generation

Concentration	Total % (SD)	Prolonged Exposure (%) SD	Exposure Stopped on GD20 (%) SD
0	97.4 (383)	91.9 (74)	98.7 (309)
100	95.4 (280)	91.7 (96)	97.2 (184)
500	91.6 (371)	30.8 (13)	93.8 (358)
1500	85.1 (215)	63.0 (54)	92.5 (161)

 

Body Weights of Pups

Concentration (ppm)	F1	F2	F3
	Day 0
0	6.1± 0.5	6.0 ±0.5	6.0 ±0.5
100	6.2 ±0.5	6.1 ±0.5	6.0 ±0.4
500	6.5 ±0.6	6.0± 0.5	6.1 ±0.6
1500	6.1± 1.0	5.7 ±0.7	5.7 ±0.2
	Day 4
0	9.7 ±0.9	9.5 ±1.4	9.7 ±1.1
100	9.8 ±0.6	10.0± 1.2	10.0 ±0.7
500	10.1 ±1.0	9.9 ±1.0	9.8 ±1.0
1500	9.2 ±1.3	9.3 ±1.0	9.2 ±0.6
	Day 7
0	13.7 ±1.3	13.3 ±1.8	14.0±2.0
100	13.2 ±1.1	13.3 ±1.6	14.1 ±1.2
500	14.0 ±1.7	13.5 ±1.4	13.4 ±1.5
1500	12.0 ±1.8	11.7 ±1.3	12.0 ±1.0
	Day 14
0	24.9± 2.7	24.3 ±2.5	26.2± 4.0
100	23.2 ±1.8	23.5 ±2.8	25.6 ±1.9
500	23.9 ±2.4	23.7 ±2.7	23.2 ±2.7
1500	19.6 ±2.7	19.3 ±1.8	20.8 ±1.3
	Day 21 Male Body Weights
0	39.5 ±5.1	40.9 ±5.5	42.9 ±7.6
100	37.2 ±5.9	39.3 ±5.5	42.7 ±3.8
500	40.0 ±4.9	39.7 ±5.6	38.7± 5.1
1500	29.9 ±3.6	30.4 ±4.2	32.8 ±3.0
	Day 21 Female Body Weights
0	38.0 ±5.0	39.6 ±5.1	41.4 ±6.2
100	35.7 ±5.7	37.9 ±4.8	41.2 ±3.6
500	38.0 ±5.0	38.6 ±5.5	37.2 ±4.8
1500	29.4 ±4.3	29.1 ±4.2	31.8 ±3.6

Applicant's summary and conclusion

Conclusions:

Concentrations of 1500 ppm (7500 mg/m3) caused reduced body weights in offspring of rats if maternal exposure was continued until delivery. If maternal exposure was ended on gestation day 20, no adverse effects were seen. The NOAEL was determined to be 1500 ppm (7500 mg/m3)

Executive summary:

Concentrations of 1500 ppm (7500 mg/m3) caused reduced body weights in offspring of rats if maternal exposure was continued until delivery. If maternal exposure was ended on gestation day 20, no adverse effects were seen. The results from the C9 aromatics represents the aromatic constituency of the C9 -C14 Aliphatics (2 -25% Aromatics). The NOAEL was determined to be 1500 ppm (7500 mg/m3).