Naphthenic acids

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with Naphthenic acids in corn oil at 100, 300 and 900 mg/kg bw. 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. The NOAEL for mating and reproductive effects was 900 mg/kg/day.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

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

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

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)

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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).

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

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

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

Reproductive effects observed:

not specified

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 pregnant^a	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+3.3^c	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+3.8^c
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 (%)

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

900 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with Naphthenic acids in corn oil (HPVIS, 2010). 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; further info on repeated & developmental parameters is given in Section 7.5.1 and 7.8.2.

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.

Extended one-generation reproductive toxicity study: a detailed justification for endpoint coverage via column 2 adaptation is added in the endpoint.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study by oral (gavage) admistration in Hannover Wistar rats was performed with Naphthenic acids in propylene glycol at the doses of 66.7, 200 and 600 mg/kg bw/day. At the highest dose, maternal toxicity was observed (clinical signs of piloerection, hunched back, reduced food intake etc.). Although lesser signs with no effect on body weight were observed at mid-dose, there were evidences of maternal toxicity. In the high dose group, the incidence of runts was higher and foetal viability was slightly lower than control. These observations were attributed to marternal toxicity. There were no such effects in the mid or lower dose groups. There were no malformations or developmental effects attributed to test item at any dose level. The following no-observed-adverse-effect level (NOAEL) were derived: 66.7 mg/kg bw/day, 600 mg/kg bw/day, 200 mg/kg bw/day and 600 mg/kg bw/day for NOAEL (maternal toxicity), NOAEL (embryotoxicity), NOAEL (foetotoxicity) and NOAEL (teratogenicity) respectively.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-20 to 2018-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 22 January 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2017787NA12
- Expiration date of the lot/batch: 31July2019
- Purity: Considered as 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≥ 70 Relative Humidity%)
- Solubility and stability of the test substance in the solvent/vehicle: test item formulation samples in the 10-220 mg/mL concentration range (using 1,2-propylene-glycol as vehicle) were proven to be stable for at least 15 days when stored at room temperature (20±5°C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the vehicle (1,2-propylene-glycol) at the appropriate concentrations according to the dose level and volume selected.

OTHER SPECIFICS: No correction for purity of the test item was applied.

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony.
- Age: 13 weeks old at mating
- Weight at study initiation: 181-242 g
- Fasting period before study: No
- Housing: Standard laboratory conditions; individual housing.
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany were used in the study (Batch number: 03018171025, Expiry date: 25 October 2020).
Nesting: Arbocel crinklets natural nesting material produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) was used in the study (Batch number: 05072170426, Expiry date: 19 April 2020).
- Diet and water (e.g. ad libitum): The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-24.5°C (target: 22 ± 3°C)
- Humidity (%): 31-59% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From 20 February 2018 to 22 March 2018

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was formulated in the vehicle (1,2-propylene-glycol) at the appropriate concentrations according to the dose level and volume selected.
- Formulations were prepared prior to administration to the animals at the appropriate frequency (not more than 6 days before use).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the dose range finding study, 1,2-propylene-glycol was selected as a suitable vehicle for this study.
- Concentration in vehicle: 10-220 mg/mL concentration range
- Concentration in vehicle: 0, 13.3, 40 and 120 mg/mL
- Treatment volume: 5 mL/kg body weight
- Expiry date: 31 May 2022

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and/or homogeneity was performed two times during the treatment period using a validated GC-FID (Gas Chromatography - Flame Ionization Detection) method. Top, middle and bottom samples were taken from the test item formulations during the first week and on the last week of treatment. Samples were taken in duplicate (1 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.

Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) to be less than 10%.

Any samples not required for analysis were discarded following acceptance of the results of the formulation analysis by the Principal Investigator and Study Director.

Details on mating procedure:

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Duration of test:

31 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

66.7 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected based on available information from a dose range finding study in pregnant Hannover Wistar rats. In this study, no signs of systemic toxicity were observed up to and including the 300 mg/kg bw/day dose level. Reduced food consumption (up to 50% below control), body weight (up to 13.3% below control), decreased body weight gain (55% below control for the treatment period) was observed in the High dose (1000 mg/kg) dams. The High dose was considered to be above the maximum tolerated dose and the 300 mg/kg/day dose level was well tolerated; hence it was estimated that a suitable High dose for the main study was 600 mg/kg/day. Based on this information, the doses of 66.7, 200 and 600 mg/kg bw/day were deemed suitable for the purpose of the study. The oral route was selected since it is a route of administration recommended by the OECD guideline No. 414 and it was considered suitable to provide the exposure required for this developmental toxicology study.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
- Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day).
- Only one general clinical observation was made on the first day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning.
- Detailed clinical observation was made only once on necropsy days (in the morning).
- The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT: Yes
- The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
- Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

WATER CONSUMPTION: No

SACRIFICE:
- Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40%, 400 mg/mL sodium pentobarbital solution; Supplier: Alfasan Nederland BV, Batch number: 1609291-03, Expiry date:
31 October 2019) administered by intramuscular injection and followed by exsanguination was used for euthanasia.

POST-MORTEM EXAMINATIONS: Yes
- The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterine weight: Yes
- Cervix weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of live foetuses: Yes
- Number of early and late embryonic death: Yes
- Number of foetal death: Yes
- Degree of resorptions: Yes
- Number and percentage of pre- and postimplantation losses: Yes

Fetal examinations:

- External examinations: Yes (all per litter). Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the appearance of the anogenital distance.
- Soft tissue examinations: Yes. For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were examined by means of a dissecting microscope.
- Skeletal examinations: Yes. For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid – ethanol/isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.
- Head examinations: No
- Other: All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.

Statistics:

In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi squared test was used for comparing group incidences against the controls.

Indices:

Percentage pre-implantation loss was calculated as:
[(number of corpora lutea-number of implantations)/number of corpora lutea] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses)/number of implantations] x 100

Sex ratio was calculated as:
% male (female) fetuses = (number of male (female) fetuses/total number of fetuses) x 100

External abnormalities/litter: %, group mean (The types and incidences of any abnormality or relevant alterations were individually listed.)
(Number of foetuses with abnormality x100)/ Number of foetuses

Visceral abnormalities/litter: %, group mean (The types and incidences of any abnormality or relevant alterations were individually listed.)
(Number of foetuses with abnormality x100) / Number of foetuses

Skeletal abnormalities/litter: %, group mean (The types and incidences of any abnormality or relevant alterations were individually listed.)
(Number of foetuses with abnormality x100)/ Number of foetuses

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Thin fur and/or alopecia on some animals were observed in all groups (Control: 5 out of 25 animals, Low, Mid and High dose groups: 2 out of 24, 25, and 25 animals, respectively). Slightly noisy respiration was observed in 1 animal out of 24 in the Low dose group (66.7 mg/kg bw/day). These changes were most probably incidental, and not considered to be test item related effects.

Piloerection was present in 1 out of 24 animals in the Low dose group (66.7 mg/kg bw/day), and all animals in the Mid and High dose groups (25 out of 25 animals, 200, and 600 mg/kg bw, respectively). Furthermore, hunched back was observed in 14 out of 25 animals in the High dose group (600 mg/kg bw/day). Two High dose animals showed red discharge from the nose, and one of these animals (animal number 4510) had decreased activity from Day 17. Based on the dose dependency and consistency of these effect in the Mid and High dose groups (200 and 600 mg/kg bw/day), they are considered to be test-item related, adverse maternal effects.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One High dose animal died on GD15 after tonic convulsion of the whole body, following a gavage accident. Due to a technical error (different cage was selected and moved to the necropsy room), one Low dose animal was preterminally euthanized.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant body weight reduction was observed in the High dose group (600 mg/kg bw/day) from GD 12 up to the end of the treatment. These changes resulted in a statistically significant reduction of body weight gain, corrected body weight gain and net body weight gain values during the treatment period (GD 6-20) and the entire study (GD 0-20) when compared to the control values.

No test item related effect on body weight was observed in the Low and the Mid dose groups (66.7, and 200 mg/kg bw/day) when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain or corrected body weight gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value, for these groups

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was statistically significantly lower between GD 6 and 10; and between GD 12 and 14 in the Mid dose group (200 mg/kg), and between GD 6 and 14 in the High dose group (600 mg/kg). Although the food consumption was normalized by the end of the treatment, food consumption for the treatment period and the entire period of the study was statistically significantly lower for these two groups. These lower food intake values were considered to be test item related effects.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Focal or multifocal thickening of the non-glandular mucosal region of the stomach was observed in 1 out of 25 Mid dose (200 mg/kg bw/day) and 12 out of 25 High dose (600 mg/kg bw/day) animals. Furthermore, one High dose animal showed red discolouration of the same region in the stomach. These findings are considered to be test item related, local adverse effects, probably an irritant effect in the stomach.

Correlated to the clinical observations in the in life phase, alopecia or thin fur was observed on some animals in all groups. However, these finding were considered as incidental and not related to the test item administration.

Perforation of the thoracic region of the oesophagus, yellow, dried material on the face of the animal (perioral), and non-collapsed lung with diffuse, dark red discoloration on all lobes were observed in the animal found dead, indicating a gavage accident, and not a test-item related death.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The maternal findings showed a clear maternal toxicity at the High dose, with clinical signs, signs of stomach irritation, and lower food intake and body weight than controls (Average body weight on GD 20 was approximately 8% below control). In the Mid dose group piloerection was observed in most animals, 1/25 animals had an indication of stomach irritation, the group had a transient lower food intake, but there was no consequence on body weight. It is concluded that there was a slight maternal toxicity at the Mid dose.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

The percentage of the early embryonic loss was significantly higher in the High dose group.
See section 'Any additional information on results' for detailed data tables.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Number of implantations showed a statistically significant decrease in the High dose group, while in the Low and Mid dose groups, the measured values were comparable to the control values.

- preimplantation loss: 9.92%, 13.88%, 15.27% and 17.65% at 0, 66.7, 200, and 600 mg/kg bw/day respectively.

- post-implantation loss: 3.82%, 2.15%, 7.87% and 8.45% at 0, 66.7, 200, and 600 mg/kg bw/day respectively.
See section 'Any additional information on results' for detailed data tables.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no total litter losses by resorption observed. See Section 'Additional information on results' for more details.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on early or late resorptions observed. See Section 'Additional information on results' for more details.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the number of dead fetuses observed.
See section 'Any additional information on results' for detailed data tables.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no total litter losses by resorption observed. See Section 'Additional information on results' for more details.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of confirmed pregnant evaluated dams was 23/24 in the Control group, 21/24 in the Low dose group (66.7 mg/kg bw/day), 24/25 in the Mid dose group (200 mg/kg bw/day) and 20/25 in the High dose group (600 mg/kg bw/day).

Originally, mating of 24 animals per groups was planned. However, one extra animal per group was mated for the Mid and High dose groups, to ensure the required number of pregnant, evaluated animals in the study.
See section 'Any additional information on results' for detailed data tables.

Other effects:

not specified

Details on maternal toxic effects:

While other statistically significant differences were found in the number of corpora lutea and total intrauterine mortality in the Mid and High dose groups, these findings were not considered to be the consequence of a test item related, adverse effect.

Although the number of corpora lutea was statistically significantly higher in the Mid dose groups, this increase is considered to be incidental, and the mean number of corpora lutea was comparable with the control in all test item treated groups. The treatment of animals started after the formation of the corpora lutea, so this difference cannot be treatment related.
See section 'Any additional information on results' for detailed data tables.

Key result

Dose descriptor:

NOAEL

Effect level:

66.7 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Remarks on result:

other: Based on significant maternal effects at 600 mg/kg and slight, local effects at 200 mg/kg bw/day.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

All of the external findings were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence; therefore, they were considered as incidental findings.
See section 'Any additional information on results' for detailed data tables.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The mean number of viable foetuses was significantly lower in the High dose group when compared to the control. The mean number of viable foetuses was comparable with the control mean in the Low and Mid dose groups.
See section 'Any additional information on results' for detailed data tables.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

There was no statistically significant difference in the sex distribution of foetuses between the control and treatment groups.
See section 'Any additional information on results' for detailed data tables.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The mean foetal weight per litter in the test item did not differ significantly from the control mean value in any treated group.
See section 'Any additional information on results' for detailed data tables.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the external findings were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings.
See section 'Any additional information on results' for detailed data tables.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the skeletal findings corresponded with the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response.
See section 'Any additional information on results' for detailed data tables.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence (in the case of convoluted ureter, which was present only in the High dose group, with a greater occurrence than in the historical control); therefore, they were considered as incidental findings.
See section 'Any additional information on results' for detailed data tables.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

While the mean early embryonic loss values of the test item treated groups were comparable with control, the percentage of the early embryonic loss was significantly higher in the High dose group. No statistically significant difference was found for the Low or Mid dose values when compared to the control.

The late embryonic loss values of the test item treated groups were comparable with control. The number of dead foetuses were 1, 0, 1, and 0 in 1, 0, 1 and 0 litters for the Control, Low, Mid and High dose groups, respectively. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups. Although total intrauterine mortality was significantly higher in the Mid and High dose groups, this statistical significance was considered to be due to the low Postimplantation loss values for the Control group in this particular study, and most probably not a toxicological effect.

There were no test item related effects on external or visceral development of foetuses in the study. In one High dose litter there was a relatively high incidence of retarded ossification at skeletal examination, this litter also had reduced foetal weights; the findings are compatible with the maternal toxicity in this group. There were no other differences observed between the groups at foetal examination.

Foetal malformations observed in the study were all considered to be incidental, they showed no dose dependency, thus not regarded as a test item related effect.
See section 'Any additional information on results' for detailed data tables.

Key result

Dose descriptor:

NOAEL

Remarks:

Foetotoxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

skeletal malformations

Remarks on result:

other: In the High dose group (600 mg/kg b/day), the incidence of runts was higher and foetal viability was slightly lower than control; also one litter had reduced foetal weight and reduced ossification; these observations were attributed to maternal toxicity.

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the lack of any test-item related intrauterine effect in any treatment group.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

Teratogenicity

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: Based on the lack of any developmental effects in any treatment group.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Detailed data tables:

Table 1: summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of mated females	24	24	25	25
Pre-terminal death or euthanasia	0	1	0	1
Number of non-pregnant females	1	2	1	4
Number of females with ≤ 5 implantation sites	0	0	0	1
Number of evaluated females on GD20 (Caesarean section)	23	21	24	20

 

Table 2: Summary of the intraunterine evaluation

 

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of evaluated dams	23	21	24	20
Mean number of corpora lutea	11.74  +/-1.48	11.76 +/-1.73	12.71* +/-1.27	11.45 +/-1.82	DN
Preimplantation loss, mean	1.22 +/-1.20	1.76 +/-2.12	1.96 +/-1.27	2.05 +/-1.79	NS
Preimplantation loss (%), mean	9.92 +/-9.58	13.88 +/-14.81	15.27 +/-9.89	17.65 +/-15.96	NS
Mean number of implantations	10.52 +/-1.38	10.00 +/-1.79	10.75 +/-1.54	9.40* +/-2.21	DN
Early embryonic loss, mean	0.26 +/-0.69	0.05 +/-0.22	0.50 +/-1.64	0.60 +/-0.75	NS
Early embryonic loss (%), mean	2.66 +/-7.42	0.60 +/-2.73	4.21 +/-13.71	6.13* +/-7.82	U
Late embryonic loss, mean	0.09 +/-0.29	0.14 +/-0.48	0.29 +/-0.91	0.20 +/-0.70	NS
Late embryonic loss (%), mean	0.72 +/-2.40	1.56 +/-5.63	3.06 +/-9.85	2.33 +/-8.52	NS
Dead foetuses, mean	0.04 +/-0.21	0.00 +/-0.00	0.04 +/-0.20	0.00 +/-0.00	NS
Dead foetuses (%), mean	0.43 +/-2.09	0.00 +/-0.00	0.60 +/-2.92	0.00 +/-0.00	NS
Postimplantation loss, mean	0.39 +/-0.78	0.19 +/-0.68	0.83 +/-1.79	0.80 +/-0.89	NS
Postimplantation loss (%), mean	3.82 +/-8.02	2.15 +/-8.27	7.87 +/-16.05	8.45 +/-10.19	NS
Total intrauterine mortality, mean	1.61 +/-1.41	1.95 +/-2.13	2.79* +/-2.11	2.85* +/-1.66	DN
Total intrauterine mortality (%), mean	13.44 +/-11.29	15.83 +/-15.93	21.89 +/-16.91	25.19* +/-14.95	DN
Viable foetuses, mean	10.13 +/-1.60	9.81 +/-1.99	9.92 +/-2.30	8.60* +/-2.30	DN

Table 2: Summary of the intrauterine evaluation

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

DN: *= p<0.05; **= p<0.01; Dunnett two-sided test

U: *= p<0.05; **= p<0.01; Dunn two-sided test

 

Table 3: Selected body weight parameters

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of evaluated dams	23	21	24	20
Body weight on GD20 (g)	317.0 +/-19.3	313.3 +/-17.5	308.8 +/-22.7	291.5** +/-27.6	U
Body weight gain GD6-20 (g)	79.0 +/-12.0	75.8 +/-12.9	67.5 +/-14.9	49.6** +/-27.9	U
Body weight gain GD0-20 (g)	98.3 +/-14.3	97.3 +/-14.5	89.5 +/-15.6	71.8** +/-26.5	U
Corrected body weight on GD20 (g)	258.8 +/-12.9	256.5 +/-12.1	252.9 +/-16.1	244.0* +/-19.8	U
Corrected body weight gain GD0-20 (g)	40.1 +/-7.8	40.5 +/-9.4	33.5 +/-11.5	24.3** +/-20.0	U
Net body weight gain GD6-20 (g)	20.8 +/-6.4	19.0 +/-5.8	11.5 +/-11.7	2.1** +/-21.2	U
Gravid Uterus (g)	58.2 +/-9.5	56.8 +/-10.0	56.0 +/-10.9	47.5** +/-12.8	U

Notes: Body weight data were rounded to one decimal place. Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

U: *= p<0.05; **= p<0.01; Dunn two-sided test.

 

Table 4: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of examined litters	23	21	24	20
Viable foetuses, mean	10.13 +/-1.60	9.81 +/-1.99	9.92 +/-2.30	8.60* +/-2.30	DN
Male foetuses, mean	5.30 +/-1.84	4.95 +/-1.91	4.38 +/-2.20	4.50 +/-1.73	NS
Female foetuses, mean	4.83 +/-1.85	4.86 +/-1.59	5.54 +/-2.11	4.10 +/-2.17	NS
Total number of foetuses	233 +/-96.3	206 +/-98.1	238 +/-92.2	172* +/-91.5	CH^2
Total number of male foetuses	122 +/-52.4	104 +/-50.5	105 +/-44.1	90 +/-52.3	NS
Total number of female foetuses	111 +/-47.6	102 +/-49.5	133 +/-55.9	82 +/-47.7	NS
Sex distribution (% of males / females)	53 / 47	50 / 50	43 / 57	55 / 45	NS
Mean foetal weight / litter (g)	3.382 +/-0.217	3.397 +/-0.181	3.291 +/-0.172	3.229 +/-0.466	NS
Number of foetuses with retarded body weight	6	4	10	25**	CH^2
Number of affected litters (with runts)	5	4	9	7	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

DN: *= p<0.05; Dunnett two-sided test

CH²: *= p<0.05; **= p<0.01; Chi Square test

 

Table 5: Summary table of the external abnormalities

Parameter			Dose (mg/kg bw/day)							HC data
			0	66.7		200		600
Total number of examined litters			23	21		24		20		670
Total number of examined foetuses			233	206		238		172		6889
Total number of intact (normal) foetuses			232	203		237		172		--
Total number of foetuses / litters with malformation			1 / 1	0 / 0		0 / 0		0/ 0		--
Total number of foetuses / litters with variation			11 / 0	3 / 2		1 / 1		0 / 0		--
External malformations
Mandible, small	Litter incidence	n	1	0		0		0		1
		%	4.3	0.0		0.0		0.0		0.1
	Foetal incidence	n	1	0		0		0		1
		%	0.429	0.000		0.000		0.000		0.015
External variations
Subcutaneous haemorrhage (neck)	Litter incidence	n	0		2		0		0	1
		%	0.0		9.5		0.0		0.0	0.1
	Foetal incidence	n	0		3		0		0	1
		%	0.000		1.456		0.000		0.000	0.015
Subcutaneous oedema, localized	Litter incidence	n	0		0		1		0	--
		%	0.0		0.0		4.2		0.0	--
	Foetal incidence	n	0		0		1		0	--
		%	0.000		0.000		0.420		0.000	--

Notes: Numbers represent the number of abnormalities/number of affected litters.

HC: historical control

No statistically significant differences were noted when compared to the control group.

 

Table 6 Summary table of the visceral abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	21	24	20	670
Total number of examined foetuses			116	103	119	86	3450
Total number of intact (normal) foetuses			114	100	115	79*	--
Total number of foetuses / litters with malformation			1 /1	0 / 0	0 / 0	1 / 1	--
Total number of foetuses / litters with variation			1 / 1	3 / 3	4 / 3	6* / 6	--
Visceral malformations
Situs inversus, total	Litter incidence	n	1	0	0	0	4
		%	4.3	0.0	0.0	0.0	0.6
	Foetal incidence	n	1	0	0	0	4
		%	0.862	0.000	0.000	0.000	0.116
Ventricular septum, defect	Litter incidence	n	0	0	0	1	1
		%	0.0	0.0	0.0	5.0	0.1
	Foetal incidence	n	0	0	0	1	1
		%	0.000	0.000	0.000	1.163	0.029
Brachiocephalic trunk, short	Litter incidence	n	0	0	1	2	45
		%	0.0	0.0	4.2	10.0	6.7
	Foetal incidence	n	0	0	1	2	53
		%	0.000	0.000	0.840	2.326	1.536
Renal papilla, absent, unilateral	Litter incidence	n	0	1	0	0	--
		%	0.0	4.8	0.0	0.0	--
	Foetal incidence	n	0	1	0	0	--
		%	0.000	0.971	0.000	0.000	--
Ureter convoluted	Litter incidence	n	0	0	0	3	7
		%	0.0	0.0	0.0	15.0	1.0
	Foetal incidence	n	0	0	0	3*	7
		%	0.000	0.000	0.000	3.488	0.203
Thymic cord	Litter incidence	n	0	1	2	1	69
		%	0.0	4.8	8.3	5.0	10.3
	Foetal incidence	n	0	1	3	1	82
		%	0.000	0.971	2.521	1.163	2.377
Renal papilla, small	Litter incidence	n	1	1	0	0	20
		%	4.3	4.8	0.0	0.0	3.0
	Foetal incidence	n	1	1	0	0	22
		%	0.862	0.971	0.000	0.000	0.638

Notes: Numbers represent the number of abnormalities/number of affected litters.

HC: historical control; n: number, %: of abnormalities

*= p<0.05; Chi Square test

 

Table 7: Summary table of the skeletal abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	21	24	20	669
Total number of examined foetuses			117	103	119	86	3435
Total number of intact (normal) foetuses			105	91	107	74	--
Total number of foetuses / litters with malformation			2 / 2	4 / 3	1 / 1	1 / 1	--
Total number of foetuses / litters with variation			10 / 9	8 / 6	11 / 8	11 / 7	--
Skeletal malformations
Skull: Mandible, short, fused	Litter incidence	n	1	0	0	0	1
		%	4.3	0.0	0.0	0.0	0.1
	Foetal incidence	n	1	0	0	0	1
		%	0.855	0.000	0.000	0.000	0.029
Scapula, short	Litter incidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetal incidence	n	0	0	1	0	--
		%	0.000	0.000	0.840	0.000	--
Transverse process fused; Pelvic girdle, malpositioned	Litter incidence	n	1	3	0	1	6
		%	4.3	14.3	0.0	5.0	0.9
	Foetal incidence	n	1	4	0	1	6
		%	0.855	3.883	0.000	1.163	0.175
Skeletal variation
Skull: 4 or More Bones, Incomplete Ossification	Litter incidence	n	3	2	1	1	123
		%	13.0	9.5	4.2	5.0	18.4
	Foetal incidence	n	4	3	1	1	153
		%	3.419	2.913	0.840	1.163	4.454
Skull: Unossified	Litter incidence	n	1	0	1	1	3
		%	4.3	0.0	4.2	5.0	0.5
	Foetal incidence	n	1	0	1	3	4
		%	0.855	0.000	0.840	3.488	0.116
Skull: Hyoid, body, unossified	Litter incidence	n	0	0	0	1	--
		%	0.0	0.0	0.0	5.0	--
	Foetal incidence	n	0	0	0	1	--
		%	0.000	0.000	0.000	1.163	--
Skull: Fontanelle Large	Litter incidence	n	0	0	1	1	3
		%	0.0	0.0	4.2	5.0	0.45
	Foetal incidence	n	0	0	1	1	3
		%	0.000	0.000	0.840	1.163	0.087

 

Sternum: Ossified Sternebra (3 or less) #	Litter incidence	n	1	2	2	3	48
		%	4.3	9.5	8.3	15.0	7.2
	Foetal incidence	n	1	2	3	6^*CH	57
		%	0.855	1.942	2.521	6.977	1.659
Sternum: Misaligned	Litter incidence	n	0	0	1	0	7
		%	0.0	0.0	4.2	0.0	1.046
	Foetal incidence	n	0	0	2	0	7
		%	0.000	0.000	1.681	0.000	0.204
Sternum: Sternebra, branched	Litter incidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetal incidence	n	0	0	1	0	--
		%	0.000	0.000	0.840	0.000	--
Sternum: Sternebra, misshapen, split	Litter incidence	n	1	0	0	0	--
		%	4.3	0.0	0.0	0.0	--
	Foetal incidence	n	1	0	0	0	--
		%	0.855	0.000	0.000	0.000	--
Ribs: Wavy, marked	Litter incidence	n	1	2	1	2	198
		%	4.3	9.5	4.2	10.0	29.6
	Foetal incidence	n	2	3	2	2	311
		%	1.709	2.913	1.681	2.326	9.054
Ribs: Rib or Cartilage Interrupted	Litter incidence	n	1	0	1	0	123
		%	4.3	0.0	4.2	0.0	18.4
	Foetal incidence	n	1	0	2	0	153
		%	0.855	0.000	1.681	0.000	4.454
Vertebrae: 3 or More Dumbbell or Asymmetric Ossification	Litter incidence	n	1	0	0	0	24
		%	4.3	0.0	0.0	0.0	3.6
	Foetal incidence	n	1	0	0	0	24
		%	0.855	0.000	0.000	0.000	0.669
Vertebrae: Sacral Unossified	Litter incidence	n	0	0	0	1	--
		%	0.0	0.0	0.0	5.0	--
	Foetal incidence	n	0	0	0	1	--
		%	0.000	0.000	0.000	1.163	--
Vertebrae: Unossified	Litter incidence	n	0	0	0	1	--
		%	0.0	0.0	0.0	5.0	--
	Foetal incidence	n	0	0	0	4^*CH	--
		%	0.000	0.000	0.000	4.651	--
Vertebrae: Dumbbell Shaped	Litter incidence	n	0	1	0	1	--
		%	0.0	4.8	0.0	5.0	--
	Foetal incidence	n	0	1	0	1	--
		%	0.000	0.971	0.000	1.163	--

 

Vertebrae: Bipartite Ossification	Litter incidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetal incidence	n	0	0	1	0	--
		%	0.000	0.000	0.840	0.000	--
Limbs (C/T): Ischium, Pubis Unossified	Litter incidence	n	0	0	0	1	7
		%	0.0	0.0	0.0	5.0	1.046
	Foetal incidence	n	0	0	0	4^*CH	7
		%	0.000	0.000	0.000	4.651	0.204
Limbs (C/T): Carpal ≤2.5	Litter incidence	n	0	0	0	2	5
		%	0.0	0.0	0.0	10.0	0.747
	Foetal incidence	n	0	0	0	5	5
		%	0.000	0.000	0.000	5.814	0.146
Limbs (C/T): Tarsal ≤3.5	Litter incidence	n	0	0	1	3	60
		%	0.0	0.0	4.2	15.0	8.968
	Foetal incidence	n	0	0	1	6	83
		%	0.000	0.000	0.840	6.977	2.416

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

CH: Chi²test

* = p < 0.05

#: Ossified sternebra (3 or less) is recorded in the historical control database

Examination of viable foetuses

The total number of retarded foetuses was significantly higher in the High dose group.

The foetal observations in the High dose group were compatible with maternal toxicity, reduced maternal body weight.

Evaluation of placentas

No abnormalities were observed on the placentas of any animals in the Control, Low (66.7 mg/kg bw/day), Mid (200 mg/kg bw/day) or High (600 mg/kg bw/day) dose groups.

Conclusions:

In conclusion, Naphthenic acid, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600 mg/kg bw/day induced maternal toxicity, with clinical signs of piloerection and hunched back, reduced food intake, reduced body weight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. The Mid dose of 200 mg/kg bw/day had lesser signs with no effect on body weight, but there was evidence of a slight maternal toxicity. In the High dose group, the incidence of runts was higher and foetal viability was slightly lower than control; also one litter had reduced foetal weight and reduced ossification; these observations were attributed to maternal toxicity. There were no such effects in the Mid or Low dose groups. There were no malformations or developmental effects attributed to test item at any dose level.


The following no-observed-adverse-effect levels (NOAEL) were derived:
NOAEL(maternal toxicity): 66.7 mg/kg bw/day
Based on significant maternal effects at 600 mg/kg and slight effects at 200 mg/kg bw/day.

NOAEL(embryotoxicity): 600 mg/kg bw/day
Based on the lack of any test-item related intrauterine effect in any treatment group.

NOAEL(foetotoxicity): 200 mg/kg bw/day
Based on the incidence of runts and foetal viability at 600 mg/kg bw/day, attributed to maternal toxicity.

NOAEL(teratogenicity): 600 mg/kg bw/day
Based on the lack of any developmental effects in any treatment group.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal development toxicity study in rats:

A developmental toxicity study was performed to assess the effects of the test item Naphthenic acid on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (1,2-propylene-glycol) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20.

The doses were selected based on available information of the chemical nature and characteristics of the test item and available results of a developmental toxicity screening test. Based on this information, the doses of 66.7, 200 and 600 mg/kg bw/day were deemed suitable for the purpose of the study.

Test item formulations were analysed for concentration and homogeneity two times during the treatment period using a validated GC-FID method. Simultaneously, vehicle control formulations were analysed for concentration.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams was 23 in the Control, 21 in the Low (66.7 mg/kg bw/day), 24 in the Mid (200 mg/kg bw/day) and 20 in the High (600 mg/kg bw/day) dose groups, respectively.

 

Results : All test item formulations were within the range of 93-104.4% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

No test item related mortality was observed in the study. Piloerection was present in all Mid and High dose animals, accompanied by hunched back in 14 out of 25 animals, red discharge from the nose in 2 out 25 animals, and decreased activity in 1 out 25 animals in the High dose groups. These adverse effects were considered to be test item related. Statistically significant lower body weight was observed in the High dose group from GD 12 up to the end of the treatment, with significantly reduced body weight gain, corrected body weight gain and net body weight gain values. These changes were considered to be test item related adverse effects.

There was no test item related adverse effect on maternal body weight / body weight gain, maternal corrected body weight / body weight gainin the Low and Mid dose groups (66.7 and 200 mg/kg bw/day, respectively).

Statistically significant reduction of the food consumption was observed in the High dose animals after the start of the treatment, a lesser transient reduction was seen in the Mid dose. Food intake was normalized by the end of the treatment, but the reduction contributed to lower body weight in the High dose group. At necropsy of the dams, no remarkable external observations related to test item treatment were recorded for any pregnant animals during necropsy.Dose-dependent increase of focal or multifocal thickening of the non-glandular mucosal region of the stomach was observed in the Mid and High dose animals, indicating a test item related adverse local effect. No remarkable abnormalities were observed on the placentas in any examined groups.

 

The maternal findings showed a clear maternal toxicity at the High dose, with clinical signs, signs of stomach irritation, and lower food intake and bodyweight than controls (Average body weight on GD 20 was approximately 8% below control). In the Mid dose group piloerection was observed in most animals, 1/25 animals had an indication of stomach irritation, the group had a transient lower food intake, but there was no consequence on body weight. It is concluded that there was a slight maternal toxicity at the Mid dose. Number of implantations showed a statistically significant decrease in the High dose group. While some intrauterine parameters showed statistically significant difference from the control values, no intrauterine parameters could be interpreted as a test item related effects. The mean number of viable foetuses was comparable with the control mean in the Low and Mid dose groups. The mean number of viable foetuses per litter was statistically lower than control, in the presence of maternal toxicity. There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups. The total number of retarded foetuses was statistical significantly higher in the High dose animals, where maternal toxicity was observed. There were no test item related effects on external or visceral development of foetuses in the study. In one High dose litter there was a relatively high incidence of retarded ossification at skeletal examination, this litter also had reduced foetal weights; the findings are compatible with the maternal toxicity in this group. There were no other differences observed between the groups at foetal examination. Foetal malformations observed in the study were all considered to be incidental, they showed no dose dependency, thus not regarded as a test item related effect.

In conclusion, Naphthenic acid,when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600 mg/kg bw/day induced maternal toxicity, with clinical signs of piloerection and hunched back, reduced food intake, reduced body weight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. The Mid dose of 200 mg/kg bw/day had lesser signs with no effect on body weight, but there was evidence of a slight maternal toxicity. In the High dose group, the incidence of runts was higher and foetal viability was slightly lower than control; also one litter had reduced foetal weight and reduced ossification; these observations were attributed to maternal toxicity. There were no such effects in the Mid or Low dose groups. There were no malformations or developmental effects attributed to test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived: for maternal toxicity 66.7 mg/kg bw/day (Based on significant maternal effects at 600 mg/kg and slight effects at 200 mg/kg bw/day), for embryotoxicity 600 mg/kg bw/day (Based on the lack of any test-item related intrauterine effect in any treatment group), for foetotoxicity 200 mg/kg bw/day (Based on the incidence of runts and foetal viability at 600 mg/kg bw/day, attributed to maternal toxicity) and for teratogenicity 600 mg/kg bw/day (Based on the lack of any developmental effects in any treatment group).

Combined repeated dose toxicity with reproduction/developmental screening :

A supporting combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in Wistar rats was performed by oral gavage with Naphthenic acids in corn oil (HPVIS, 2010). 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 developmental toxicity parameters are discussed. Treatment of Sprague-Dawley rats with refined naphthenic acids had no apparent effects on mating and did not produce malformations at the highest dose tested (900 mg/kg/day). There were significant reductions in number of offspring, number live born and offspring body weights at 300 and 900 mg/kg, which were considered secondary findings at the maternally toxicity dose levels.

 

Justification for classification or non-classification

Available data suggest values that warrant no classification for reproductive and developmental toxicity under Regulation 1272/2008 (CLP).