Naphthenic acids, reaction products with diethylenetriamine

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 06 - August 26, 2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity screening

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, reaction products with diethylenetriamine

EC Number:

268-610-1

EC Name:

Naphthenic acids, reaction products with diethylenetriamine

Cas Number:

68131-13-5

Molecular formula:

C16H21N3O2 (minimal)

IUPAC Name:

Naphthenic acids, reaction products with diethylenetriamine

Details on test material:

Name: MK195KSF-Naphthenic acids, reaction products with diethylenetriamine
Physical state at RT: Hazy solid
Colour: orange/brown
Volatile: No
Density: 0.83
pH: 9.07
Storage Conditions: at room temperature
Safety Precautions: Routine hygienic procedures will be sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young healthy male and nulliparous, non pregnant female rats [strain: Wistar Crl:WI] (Full-Barrier), were used in this study. The animals were derived from a controlled full barrier maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
At the beginning of the study, the age of the animals was 8-9 weeks. The range of the body weight was:
Females: 154 to 183 g, (mean: 167.93 g, ± 20%= 33.59 g)
Males: 213 to 233 g, (mean: 222.30 g, ± 20%= 44.46 g)
Housing and Feeding Conditions
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full barrier) in air-conditioned rooms under the following conditions:
Temperature: 22 3 °C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no.: xx).
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed at BSL BIOSERVICE

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
Name: Corn oil
Batch No.: 11KBC6753
Storage conditions: at room temperature (RT),
Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety

Details on mating procedure:

Animals were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of female were checked to confirm the evidence of mating. The day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-30 days.The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-30 days.

Frequency of treatment:

daily; the animals were dosed with the test item on 7 days per week basis.

Details on study schedule:

After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.
Males and females were sacrificed on treatment day 29-31, post natal day 4 respectively and subjected to necropsy. Non Pregnant females were sacrificed on their respective day 26 after the evidence of mating.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

80 animals (10 females and 10 males/group) were included in the study

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Animals were examined for the daily clinical signs, mortality, weekly detailed clinical observations, pre treatment and at termination for functional observations. Body weight and food consumption was measured weekly except food consumption was not measured during the mating period (female) and mating/post mating period (male). Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Litter observations:

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Results and discussion

Results: P0 (first parental generation)

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Test item related clinical findings viz., piloerection, vocalization, pushing the bedding and salivation were recorded in most animals of treatment groups during the study. Occasionally findings like reduced spontaneous activity, weight loss, dyspnoea, diarrhoea, epistaxis, nasal discharge, dehydration, etc were also observed.

During the study twelve rats were found dead or sacrificed moribund. Out of these, 1 male from control, 1 male and 1 female from LD group and 3 males/ 6 females of HD group died. The death of animals were histopathologically termed possibly due to misgavaging except for three dead females (animal no.s 34, 35, 39) in HD group, whose death could not be determined due to advanced autolysis.

In males, the statistical analysis revealed significantly low body weight development during premating period in HD group and during first week of mating/post mating period in MD group, which further recovered with the progress of the study. But, overall the body weight development was significantly lower in MD and HD group. In females, significantly low body weight development were observed during second week of premating period in HD group and during GD 0-7 (HD group), GD 14-20 (MD group), GD 14-20 (HD group). There was also a consistent dose response pattern observed for all treatment groups when compared to the corresponding controls.

In males and females, statistical analysis reavelaed significantly low food consumption during premating period in HD group when compared to the corresponding controls. In females, significantly low food consumption was also recorded during GD 0-7 and 14-20 in MD and HD groups when compared to corresponding controls.

The litter weight data revealed statistically significant deviation for number of female pups, total number of pups, total litter weight (PND 0 and 4), female litter weight (PND 0 and 4) in HD group when compared with corresponding controls.

The groups mean litter weight (PND 4) also revealed statistically significant deviation in MD group when compared to the control. But, there was no dose response pattern observed.

Statistically significant difference was observed for precoital interval and duration of gestation in the females of HD group when compared to corresponding controls. All pregnancies resulted in normal births except for one animal (animal no. 37) in HD group, which resulted in still births.

Successful mating resulted in 10/10 pregnancies in control, 9/9 pregnancies in LD groups, 9/10 pregnancies in MD groups and 3/5 pregnancies in HD. The fertility index observed were as follows, Control- 100%; LD- 100%; MD groups (90%) and HD (60 %). Significantly low fertility index and delivery index was recorded in HD group when compared to corresponding controls.

The statistically significant low number of implantation sites, number of live pups and significantly high rate of percent pre and post implantation were recorded in HD group when compared to corresponding control.

The statistically significant low total pups born, live pups (PND 0 and 4) and significantly high number of still births were recorded in HD group when compared to the corresponding controls.

Survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups. Gross necropsy of dead pups, if any, revealed no treatment related findings.

Statistically significant deviation was observed for few hematological parameters viz., WBC, MCV, MCH, platelet, PT, Neutrophil, lymphocytes and monocytes in MD or HD groups when compared to corresponding control values.

Statistically significant deviation was obsereved for few clinical biochemistry parameters viz., ALBB, SGPT and Na in LD or MD or HD groups when compared to corresponding control values.

The urinalysis revealed no significant differences in any of the treatment groups compared to corresponding control.

In males, the macroscopic findings observed were as follows, Control- small testes (1/10), small epididymides (1/10), discoloured axillary lymph node (1/10), discoloured thymus (1/10), small spleen (1/10), large and discoloured lung (1/10); LD- discoloure lung (1/10), enlarged mesenteric lymph node (1/10); MD- discoloured axillary lymph node (1/10), discoloured thymus (1/10), lung with small black spots (1/10); HD- small testes (2/10), small epididymides (1/10), small spleen (2/10), enlarged lung (2/10), discoloured lung (2/10), small Seminal vesicle+coagulating gland (2/10), discoloured liver (2/10), discoloured pancrea (1/10), gas filled stomach and intestine (2/10), small thymus (2/10), foamy nasal discharge (1/10).

In females, control- cyst on ovary (1/10); LD- cyst on ovary (1/10), discoloured intestine (1/10); MD- bloody lung (1/10); HD- bloody lung (2/7), gas filled stomach and intestine (3/7), small spleen (1/7), small liver (1/7), small thymus (1/7), dilated intestine (1/7), enlarged mesenteric lymph node (2/7), discoloured Kidney (2/7).

In males, statistically significant low mean values were observed for absolute weight of heart (MD and HD groups), thymus (HD group) and seminal vesicle + coagulating gland (HD group) when compared with the corresponding controls. But, the relative weight of liver indicated significantly high values for MD and HD groups compared to control. The relative organ weight was significantly deviated for spleen (HD group), testes (MD group), thymus (MD group), Seminal vesicles + coagulating gland (HD group).

In females, statistically significant low values were observed for absolute weight of heart (LD and HD groups) and uterus with cervix (HD group); significantly high value for spleen (MD group). The relative organ weight was significantly higher for liver (HD group), low for heart (LD and HD groups) and uterus with cervix (HD group).

At terminal sacrifice, histopathological changes considered to be directly test item-related and toxicologically relevant were seen in the small intestine (jejunum and ileum), mesenteric lymph node, spleen, lung and adrenal gland in LD or MD or HD group animals. The histopathological organ changes observed in the spleen, lung, liver, bone marrow and thymus were considered secondary to a general inflammatory state caused by test item effects in treated animals, or to stress.

No effects on reproductive organs were noted on terminal sacrifice animals, the minor changes observed histopathologically were not considered to be directly test item-related.

The results of the concentration measurements revealed recoveries between 85.3 % and 98.5% in HD group, between 86.4 % and 97.2 % in MD group and between 87.6 % and 95.5 % in LD group. Homogeneous distribution of the test substance in the preparation was approved.

Applicant's summary and conclusion

Conclusions:

Based on the data generated from this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with MK 195KSF- Naphthenic acids, reaction products with diethylenetriamine, no NOAEL (No Observed Adverse Effect Level) could be established for general toxicity. However, for reproductive/ developmental toxicity, the NOAEL is believed to be 300 mg/kg bw.