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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose & reproductive/developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given bij HPVIS.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
other: robust study summary
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Naphthenic acids
EC Number:
215-662-8
EC Name:
Naphthenic acids
Cas Number:
1338-24-5
Molecular formula:
For the acidic (naphthenic) fraction: CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc. For the non-acidic fraction: not applicable
IUPAC Name:
Naphthenic acids
Test material form:
other: liquid
Details on test material:
The test sample used in the current program was a blend of naphthenic acids from three sources. The samples were dried under a stream of nitrogen and then re-dissolved in 0.5 mL dichloromethane. The samples were analyzed by GC-MS (Young et al., 2008) and the total ion current mass spectra were collected and tablulated (Holowenko et al., 2002).

Based on these data it was determined that there were no significant differences among these samples (Fedorak, 2009). The data indicated that the test material contained constituents with carbon numbers predominantly in the range of C6-C16 (corresponding to a molecular weight range of approximately 116-250) and with a ring distribution of approximately 0 rings (24%), 1 ring (39%), 2 rings (31%), 3 rings (5%) and 4 rings (1%).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 ml/kg doses.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days. All females confirmed to have mated were placed in plastic maternity cages once mating was confirmed.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Females for which copulation was not detected were placed in maternity cages at the end of the 1
- Any other deviations from standard protocol:Length of gestation was calculated as the time from confirmation of mating to the onset of delivery.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Dosing of males was initiated 14 days prior to pairing and throughout a 14 day mating period for a total of 28-29 doses. Dosing of females was also initiated 14 days prior to pairing and continued throughout the mating and gestational periods until study termination on post-natal day 3. The total number of doses ranged from 39-53 depending on the time at which mating occurred.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
900 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:Detailed physical examinations of all animals were conducted weekly (See Section 7.5.1)

BODY WEIGHT: Yes
- Time schedule for examinations: females: recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination.

FOOD CONSUMPTION:
- Food consumption by adult animals was also recorded on the same schedule as the body weights.

WATER CONSUMPTION: No

OTHER:
- Parental mating, fertility, conception and copulation indices , gestation length, numbers of former implantation sites, absolute and relative organ weights, and pre-coital intervals.
- Toxicological parameters: See Section 7.5.1.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
On the day of parturition, all pups were examined for viability, for the presence of gross malformations and to assess gender. The numbers of live and stillborn pups were recorded.
All offspring were uniquely identified and examined daily for signs of mortality and ill health. All offspring were individually weighed on PND 1 and 4. Gender was assessed on PND 0 and 4. At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.

GROSS EXAMINATION OF DEAD PUPS: no data


Postmortem examinations (parental animals):
SACRIFICE
- Male animals: 14 days after mating
- Maternal animals: . Females for which there was no evidence of mating were sacrificed on post-cohabitation day 25, those that showed evidence of mating but failed to deliver were euthanized on post-mating day 25, and all others were euthanized on post-natal day 4.

GROSS NECROPSY: YES
- At termination rats were euthanized by carbon dioxide inhalation.
- Necropsies were conducted on all animals sacrificed in extremis or at study termination.

HISTOPATHOLOGY / ORGAN WEIGHTS
- An examination of target organs including male and female reproductive organs was also carried out as part of this test (See also Section 7.5.1).
- Organs examined included: ovaries with oviduct, uterus with cervix and vagina, testes with epididymides, prostate and seminal vesicles.
- The ovaries, testes and uteri were weighed and all were examined histologically
Postmortem examinations (offspring):
All offspring were individually weighed on PND 1 and 4.
Gender was assessed on PND 0 and 4At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.
Statistics:
Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates’ correction (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, numbers of corpora lutea, number of pups born, live litter size on PND 0, unaccounted for sites, absolute and relative organ weights, and pre-coital intervals were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.
Reproductive indices:
See Tables below and in Section 7.8.2.
Mating, fertility, pregnancy and gestation indices were not provided as such, however No. of females mated, pregnant and with litters were given.
Pre-implantation loss not provided, but No. of corpora lutea and No. of implantation sites given.
Implanation index not provided, but No. of implanatation given.
Post-implantation indexes given.
Offspring viability indices:
Viability index not provided, but No. born and alive on day 4 given.
Sex ratio given (See Section 7.8.2)

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:
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)

The absolute epididymal weights were increased in the 900 mg/kg/day group but were not significantly different when expressed on a per body weight basis. The uterine weights were also significantly elevated but this was considered to have been a consequence of the fact that the females were all in lactational anaestrous. The uterine weights were within the historical range of the laboratory and were not considered to have been toxicologically important. There were no weight differences in any of the other organs and no pathological changes in any of the reproductive organs at the highest dose tested (900 mg/kg/day).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mating index
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive organ effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Decrease No. born pups at 900 mg/kg bw - See Section 7.8.2
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decrease at 900 mg/kg bw - See Section 7.8.2
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: not applicable, no adverse effects observed up to the highest dose level tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Summary of reproductive parameters assessed in the repeated dose/reproductive toxicity study of refined naphthenic acids.

Dose (mg/kg/day)

Corn Oil Control

100 mg/kg/day

300 mg/kg/day

900 mg/kg/day

 

 

 

 

 

Number of females paired

12

12

12

12

Number of female mated

12

12

10

11

Number of females pregnanta

9

12

10

11

Number of females with litters

9

12

10

11

Pre-coital interval (days)b

1.4+0.7

2.3+1.1

4.2+3.3c

3.8+3.5

Gestation length (days)

21.4+0.6

21.9+0.3

22.0+0.5

22.1+0.5

Corpora lutea

15.6+2.3

14.0+1.4

15.1+3.0

13.8+2.1

Implantation sites

15.0+2.4

13.6+1.1

13.0+1.2

12.2+3.7

Number born

14.1+1.9

12.9+1.1

12.0+1.6

10.8+3.8c

Post-Implantation loss (%)d

6.0

5.1

7.7

11.5

a. Pregnant = uterine implantation sites.

b. Data summarized as mean+standard deviation.

c. p < 0.05

d. Post-implantatoin loss = (No. of implantations - No. of life fetuses)/No. of implanatations (%)

Applicant's summary and conclusion

Conclusions:
No reproductive effects were identified. The NOAEL for mating and reproductive effects of is 900 mg/kg/day.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar ratswas performed by oral gavage with Naphtenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only reproductive toxicity parameters are discussed: (furher info on repeated & developmental parameters is given in Section 7.5.1 and 7.8.2. Target organ findings were identified at the dose of 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity.

No reproductive effects were identified up to 900 mg/kg bw . There were no weight differences in any of the other organs nor any pathological changes in the reproductive organs up to the highest dose tested (900 mg/kg/day). The NOAEL for mating and reproductive organ effects was 900 mg/kg/day.