Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Principles of method if other than guideline:
30 rats of each sex (F0 generation) were exposed via inhalation to 100, 500, or 1500 ppm of high flash aromatic naphtha for 6 hrs/day, 5 days/week for 10 weeks. Rats were then mated, and females confirmed mated were then exposed for 6 hrs/day during gestation days 0 to 20. Dams were not exposed during delivery, but exposure was reinitiated on postnatal day 5 and continued through postnatal day 21 (weaning). This F1 generation was then exposed for 10 weeks starting at 5 to 7 weeks of age, then mated to produce the F2 generation. The F2 generation was treated similarly to the F1 generation, except they were exposed immediately after weaning to produce the F3 generation. Fertility indices were calculated for all parental generations (F0, F1, and F2), female mating index, female conception index, female gestation index, male fertility index, cohabitation time, and litter size at birth. The gestational survival and postnatal survival indexes of the F1, F2, and F3 generations were also calculated.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 Naphtha, 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 Naphtha, Type 1

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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: Individually 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
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 separate 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 500, 1500 ppm
Basis:
nominal conc.
0, 500, 2500, 7500 mg/m3
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 epididymus, lung, ovary, pituitary, prostate, seminal vesicle, testis, uterus, vagina, lymph nodes were examined. Masses and gross lesions were examined from all groups.

ORGAN WEIGHTS
Epididymus, 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 animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

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)

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)
Length of time for mating was increased, but not significantly.

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. All other animals survived.

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)

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
other: 7500 mg/m3; When maternal exposure ceased before gestation day 20, there were no negative effects on fertility or on the offspring.
Remarks on result:
other: Generation: offspring of all generations (migrated information)
Dose descriptor:
LOAEC
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
other: 7500 mg/m3; Maternal lethality, and reduced body weight in offspring were noted if maternal exposure was continued beyond GD20 and until delivery. ("prolonged exposure" group in Table).
Remarks on result:
other: Generation: offspring of all generations (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
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 lactational 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 liveborn/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 weight 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:
The test substance NOAEC for fertility effects is 1500 ppm (7500 mg/m3) for male and female rats.
Executive summary:

This study was conducted to determine the reproductive toxicity of high flash aromatic naphtha. 30 rats of each sex (F0 generation) were exposed via inhalation to 100, 500, or 1500 ppm (0, 500, 2500, 7500 mg/m3) of high flash aromatic naphtha for 6 hrs/day, 5 days/week for 10 weeks. Rats were then mated, and females confirmed mated were then exposed for 6 hrs/day during gestation days 0 to 20. Dams were not exposed during delivery, but exposure was reinitiated on postnatal day 5 and continued through postnatal day 21 (weaning). This F1 generation was then exposed for 10 weeks starting at 5 to 7 weeks of age, then mated to produce the F2 generation. The F2 generation was treated similarly to the F1 generation, except they were exposed immediately after weaning to produce the F3 generation. Under these conditions, reduced survival and body weight gains were observed in the offspring of the high exposure group. Although this was evidence of a toxic effect at the highest dose tested, there were no reproductive effects.

 

Fertility indices were calculated for all parental generations (F0, F1, and F2), female mating index, female conception index, female gestation index, male fertility index, cohabitation time, and litter size at birth. The gestational survival and postnatal survival indexes of the F1, F2, and F3 generations were also calculated. The results of the fertility study show no exposure related adverse effects to fertility in either male or female rats, therefore the NOAEC for fertility is 1500 ppm (7500 mg/m3) for high flash aromatic naphtha.