Carboxylic acids, di-, C4-6

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

13-week repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Specific details on test material used for the study:

test item: AGS-Mixture (consisting of adipic acid: 3.9%; Glutaric acid: 16.43%; succinic acid 4.77% and nitric acid 3.9%)

Species:

rat

Strain:

other: Charles River CD rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage Michigan, USA
- Acclimatization period: pretest period of 2 weeks
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 163-202g, females: 131-164g
- Fasting period before study: none
- Housing: individually housed in wire-mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light/dark

IN-LIFE DATES: From August 15, 1983 : To: November 15, 1983

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Amount of vehicle (if gavage): 10 mL of the proper solution provided the correct dose (30, 100, and 300 mg test article per mL vehicle)

Analytical verification of doses or concentrations:

yes

Remarks:

dosing solution analysis: at IRDC (International Research & Development Corporation, Michigan, USA)

Details on analytical verification of doses or concentrations:

Test solutions were delivered to the Analytical Chemistry Department for sampling at the following times: priinitiation (6 days and 5 days prior to study initiation) and study weeks 1, 4, 8, and 12. Samples for evaluating stability (24 hours and 10 days following preparation) were collected from the preinitiation and week 1 preparations. The size of samples varied between 2 and 40 mL. The samples were analysed in duplicate.
The mean concentration of all the analyzed solutions ranged from 102 to 105% of the desired levels.
Studies conducted on solutions after being held for 24 hours and 10 days at normal laboratory conditions indicated that the test article was stable in solution for these intervals.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days per week

Dose / conc.:

3 other: %

Remarks:

3% of AGS mixture in 10 mL water/kg bw (calculated dose: 87 mg/kg bw/day)

Dose / conc.:

10 other: %

Remarks:

10% of AGS mixture in 10 mL water/kg bw (calculated dose: 290 mg/kg bw/day)

Dose / conc.:

30 other: %

Remarks:

30% of AGS mixture in 10 mL water/kg bw (calculated dose: 870 mg/kg bw/day)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose selection was based on a 4-week oral range finding study in rats (Monsanto Study No. IR-83-141, 1983)
- Rationale for animal assignment (if not random): computerized random selection
- Fasting period before blood sampling for clinical biochemistry: 24 hours

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, beginning with the pretest period

FOOD CONSUMPTION: yes
- Food consumption for each animal determined: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all rats during the pretest period and for all surivors during study week 13 (day 85); binocular indirect ophthalmoscope following pupillary dilatation with 1% tropicamide solution

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- How many animals: 10/sex/group
- Parameters examined:
total leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelet count, reticulocyte count, differential leucocyte count; the hematological indices mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch) and mean corpuscular hemoglobin concentration (mchc) were automatically calculated by the analyser (Ortho ELT-8)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters examined.:
AST, ALT, alkaline phophatase, Glucose, urea nitrogen, total bilirubin, total cholesterol, almumin, globulin (calculated), total protein, ceatinine, Na, K, Cl, Ca, inorganic phosphorus, ornithine carbamoyl-transferase, gamma glutamyl transpeptidase, creatine phosphokinase

URINALYSIS: Yes
- Time schedule for collection of urine:
- Animals fasted: Yes / No / Not specified
- Parameters examined:
volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrites, bilirubin, occult blood, microscopy of spun deposit

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
unscheduled necropsy: all animals dying on study
terminal necropsy: all survivors at 13 weeks
organ weights: 13 week sacrifice (scheduled): liver, kidneys, heart, adrenals, ovaries, testes, brain

HISTOPATHOLOGY: Yes
on all animals dying on study and on all animals in the control and 30% group sacrificed at study termination; all tissue masses and gross lesions examined on all animals, liver kidney and lung tissue of animals in 3 and 10% groups were examined.
Tissues processed:
adrenals, bone (femur), bone marrow (femur), brain (3 levels: fore, mid and hind brain), eyes, gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), gonads (ovaries, testes with epididymides), heart, kidneys, liver, lung with mainstem bronchi, lymph nodes (mediastinal, mesenteric and regional, when applicable), mammary region (females only), pancreas, pituitary, prostate and seminal vesicles, salivary gland, mandibular with submandibular lymph node, sciatic nere, skin, spinal cord (cervical, midthoracic and lumbar), spleen, thymic region, thyroid-parathyroid comples, trachea, urinary bladder, uterus

Statistics:

Body weight, food consumption, hematology, biochemical, urinalysis and organ weight (absolute and relative to body and brain weights) data analyzed using Bartlett’s test and the analysis of variance (one-way classification). Treatment groups compared to the control group, by sex, using the appropriate t-statistic (equal of unequal variance) and Dunnett’s multiple comparison tables. Nonparametric analyses were performed by using rank transformations on chloride, total bilirubin, ornithine carbamyltransferase, gamma glutamyltranspeptidase, and specific gravity.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related signs observed were rales, labored breathing, excessive salivation and decreased defecation. The incidence of these signs was highest in the high dose (30% AGS Mixture) group with generally more males than females affected. For the control, 3, 10 and 30% AGS Mixture does groups, rales was observed for 0/15, 2/15, 1/15, 7/15 males and 0/15, 0/15, 2/15, 2/15 females; labored breathing was observed for 0/15, 1/15, 3/15, 6/15 males and 0/15, 0/15, 0/15, 4/15 females. Excessive salivation was noted for 2 male and 1 female high dose (30% AGS Mixture) rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two high dose (30% AGS Mixture) males died, one each on study days 41 and 64 and one high dose (30% AGS Mixture) female died on study day 17. An additional high dose (30% AGS Mixture) male died just prior to the terminal sacrifice on day 92.
On the basis of macroscopic and microscopic pathology, the mode of death in one male rat was ascertained to be gavage injury. Mechanisms of death for the other rats were unknown.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases (p<0.01 or p<0.05) in mean body weight were noted for high dose (30% AGS Mixture) males at weeks 7 and 9-13. By week 13, the mean for this sex group was 10.1% less than the control mean. The slight decreases for females were not statistically significant.

Description (incidence and severity):

In males, there was a slight reduction in food consumption (g/animals/day and g/kg/day) at the mid (10% AGS Mixture) and high (30% AGS Mixture) dosage levels. Statistically significant differences from control values were apparent in both male groups; however these differences occurred to a greater degree and more frequently at the high dosage level. In females, food consumption values generally were comparable to controls. No statistically significant differences from controls were seen in the treated females except on one occasion (week 3) at the high (30% AGS Mixture) dosage level.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Observations noted for control and treated rats were considered representative of pathology that would be expected for these animals given age, sex and strain; no obvious trends in pathology suggestive of compound-related reactions were noted.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant increase in leukocytes in the 30% AGS mixture males at the 13-week interval. Segmented neutrophils and lymphocytes were both slightly elevated when compared to controls although neither were statistically significant. The significance of this finding is unknown.
There were no other test article related hematological changes seen. Statistical significance was noted for a few parameters when comparing the treated and control groups, however the values were within the normal range of historical control values for rats in this laboratory. (see attachment)

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test article related biochemical changes at the 13-week interval. Statistical significance was noted for a few parameters when comparing the treated and control groups; however the values were within the normal range of historical control values for rats in this laboratory. (see attachment)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant decrease in the urinary pH in both males and females in the 30% AGS mixture group and in males in the 10% AGS mixture group at the 13-week interval (see Table 1). All other values were within the normal range of historical control values for rats in this laboratory.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test article related organ weight variation occurred in this study.
A decrease in mean brain weight which was observed in male rats from the 30% AGS mixture group (p< 0.05) probably occurred as a result of the decreased body weight gain in this group. The brain/body weight ratio was not altered.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test article related macroscopic changes were observed among any of the male or female rats which were sacrificed at termination or died during the course of the study.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article related changes were observed in any of the tissues from male or female rats evaluated histopathologically from the high dosage group. All microscopic changes seen were considered spontaneous or incidental in nature.
A frequent lesion observed in this study was interstitial pneumonia. However, prevalence of this condition was similar in the control and treatment groups and no article effect was evident. (see attachment)

Dose descriptor:

NOEL

Effect level:

3 other: % test compound in 10 ml application volume

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponds to approx. 87 mg dicarboxylic acid/kg body weight; effects at higher doses: decrease in urinary pH; slight reduction in body weight

Critical effects observed:

no

Table 1: pH values in urine 

	control	3% AGS	10% AGS	30% AGS
males	8	8	7*	6**
females	8	8	7	5**

* p < 0.05
* p < 0.01

Executive summary:

In a sub-chronic study, conducted similar to OECD TG 408 and under GLP, the test item AGS-Mixture (consisting of adipic acid: 3.9%; Glutaric acid: 16.43%; succinic acid 4.77% and nitric acid 3.9%) was administered daily by oral gavage to 15 male and 15 female CD rats at dose levels of 3, 10 and 30% (about 87, 290 and 870 mg/kg bw/day). The vehicle used was deionized water and the dosing volume 10 ml/kg.

There were no test article related effects seen in any of the parameters evaluated for the rats receiving 3% AGS mixture orally for 13 weeks. Accordingly, this is the no effect level (NOEL) for this study.

At the mid dose level (10% AGS mixture), a few slight effects were seen. There was a slightly higher incidence of labored breathing and/or rales at this level, and while this is not uncommon in a gavage study, the incidence was even greater in the 30% AGS mixture groups suggesting that this might be a test article related effect. In addition, the females had a slightly decreased body weight gain although statistical significances was not achieved. The mid dose males had decreased urinary pH values. All other criteria evaluated were considered to be within normal limits.

At the high dose level (30% AGS mixture), definite test article related effects were seen. Three high dose males and one high dose female died during the study. Apart from one animal which died as a result of a gavage injury, no cause of death could be determined for the other two animal. No mortalities occurred at the lower dose levels or in the control animals. Mean body weights at 30% AGS mixture were significantly decreased in males; at 13 weeks the mean value was 10.1% lower than the mean weight of the control males. In females, the difference from controls at 13 weeks was 5.5% although statistical significance was not achieved. Correspondingly, food consumption (g/animal) was decreased in males. There was an increased incidence in labored breathing and/or rales in both males and females. The 30% AGS mixture males also had a statistically significant increase in leukocytes at the 13-week interval; the significance of this finding, by itself, is unknown. Both males and females had a statistically significant decrease in urinary pH at 13 weeks. There were no findings in the macroscopic or microscopic examination of tissues indicative of a test article effect or correlation with the previously mentioned findings in this group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Principles of method if other than guideline:

13-week repeated dose toxicity study with examination of reproductive organs

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C4-6

EC Number:

271-678-5

EC Name:

Carboxylic acids, di-, C4-6

Cas Number:

68603-87-2

Molecular formula:

C5H8O4, C4H6O4, C6H10O4

IUPAC Name:

Carboxylic acids, C4-6 di-

Details on test material:

AGS Mixture: lot No.5/9/83 PENSACOLA: adipic acid: 3.9%; Glutaric acid: 16.43%; succinic acid 4.77% and nitric acid 3.9%

Test animals

Species:

rat

Strain:

other: CD-rats

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 other: %

Doses / concentrations 2

Dose / conc.:

10 other: %

Doses / concentrations 3

Dose / conc.:

3 other: %

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

see chapter 7.5.1 of IUCLID (repeated dose toxicity)

Results and discussion

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

A sub-chronic study (Monsanto, 1985) was conducted similar to OECD TG 408 and performed under GLP. The test item AGS-Mixture (adipic acid: 3.9%; Glutaric acid: 16.43%; succinic acid 4.77% and nitric acid 3.9%) was administered daily by oral gavage to 15 male and 15 female CD rats at dose levels of 3, 10 and 30%. In this study examination of testes and ovary weight as well as histopathological examination of gonads (ovaries, testes with epididymides), mammary region (females only), prostate, seminal vesicles and uterus did not reveal substance-related adverse effects.

Applicant's summary and conclusion

Executive summary:

A sub-chronic study (Monsanto, 1985) was conducted similar to OECD TG 408 and performed under GLP. The test item AGS-Mixture (adipic acid: 3.9%; Glutaric acid: 16.43%; succinic acid 4.77% and nitric acid 3.9%) was administered daily by oral gavage to 15 male and 15 female CD rats at dose levels of 3, 10 and 30%. In this study examination of testes and ovary weight as well as histopathological examination of gonads (ovaries, testes with epididymides), mammary region (females only), prostate, seminal vesicles and uterus did not reveal substance-related adverse effects.