Propanoic acid, 2-hydroxy-2-methyl-, 2-propen-1-yl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Amendment No. 1 dated October 20, 1999
The contractig of histological processing of animal and organs to
BS&S (Scotland) Ltd was detailsed. A contract, which is part of the raw data set, was signed October 20, 1999.

Addionally to organ weights relative to carcass weight also organ to brain weight ratios were calculated.

For technical reasoons no food efficiency values were calculated.

The company SEAG Software Engineering AG Basel, Switzerland (PATHDATA Pathology sftware) was invloved in a merger and was renamed to SYSECA (Schweiz) AG.

The reponsible scientist for histological processing at BS&S was Mr J Smart.

The above mentioned deviations are considered to have no impact on the validity of the study. There were no circumstances that could have affected the quality and/or integrity of the data.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

EC Number:

700-908-3

Cas Number:

19444-21-4

Molecular formula:

C7H12O3

IUPAC Name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, CH-4414, Füllinsdorf, Switzerland
- Age at study initiation: Approximately 4 weeks (at delivery).
- Weight at study initiation:
- Fasting period before study:
- Housing: Individually.
- Diet : Ad libitum (except for fasting overnight prior to blood collection) - Pelleted, certified diet
- Water: Ad libitum - tap water
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 16 - 20 / hour
- Photoperiod (hrs dark / hrs light): 12/12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC, 0.1% Tween 80 in water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was administered orally by gavage (rubber catheter). The dosing volume was adjusted daily to individual body weights. The control animals were dosed with the vehicle only.

Emulsions of the test item in the vehicle at the appropriate concentrations were freshly prepare every day immediately prior to dosing.
Volume applied (ml/kg body weight): 10
Vehicle: Distilled water
0.5 % carboxymethylcellulose (CMC)
0.1 % Tween 80

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of the study, samples of the vehicle containing the test item at concentrations of 0.5, 1.5 and 15 mg/ml were analysed for content, homogeneity and stability (4 hrs at room temperature).
During treatment, control analyses of the test item concentration in the vehicle were carried out at all dose levels on samples collected once per experimental week. The samples were collected on completion of dosing, immediately deep-frozen and sent to the analytical laboratory.

Duration of treatment / exposure:

8 weeks (4 weeks daily gavage, 4 week recovery period)

Frequency of treatment:

Daily by gavage

Doses / concentrations

Remarks:

Doses / Concentrations:
0.5, 1.5 and 15 mg/ml
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Rationale for dose selection:
5 mg/kg body weight/day: This dose was expected to cause no observable effects
15 mg/kg body weight/day: This dose was expected to cause no or minimal observable effects.
50 mg/kg body weight/day: This dose was expected to cause slight effects, if any.
150 mg/kg body weight/day: This dose was expected to cause observable effects, but no or few fatalities to permit meaningful evaluation of the study.

- Rationale for animal assignment : Random

Examinations

Observations and examinations performed and frequency:

A total of 80 animals was used for the experiment: 5 males and 5 females per dose group (experimental group A), and an additional 5 males and 5 females in the control, upper mid dose and high dose groups for recovery evaluation (experiment group B).
Refer to Table 1

Schedule of Investigations:
Mortality Twice daily
Clinical signs Once daily (preferably during morning hours
Detailed clinical observations: During pre-test an once weekly thereafter
Body weight: Once weekly
Food consumption: Once weekly
Water consumption: Once weekly
Functional observational battery: Weeks 4 and 8
Motor activity: Weeks 4 and 8
Laboratory investigations: Weeks 4 and 8
Necropsy: Weeks 5 and 9

Sacrifice and pathology:

Macroscopical findings:
Macroscopical verification of the gross lesions recorded at necropsy revealed a relationship to the treatment for the following macroscopical findings:
Liver:
Foci in 2/5 males of dose group 5, experimental group A, and granulated/nodular surface in 1/5 females of dose group 4, experimental group A, and 3/5 males and 3/5 females of dose group 5, experimental group B were found in correlation to the treatment-related microscopical finding cholangiofibrosis.
Stomach:
Mucosal defect in 1/5 males dose group 5, experimental group A, was put in correlation to a Treatment related ulceration of the cutaneous membrane of the forestomach.
Small intestine:
Black-brown contents in 1/5 males of dose group 5, experimental group A, were arising from a stomach ulcer that found to be treatment related.
Fluid contents in 1/5 females of dose group 3, experimental group A, were correlated to chronic inflammation considered treatment related.

Large intestine:
Foci and reddish discoloration in 1/5 males of dose group 5, experimental group A, were correlated to a treatment related chronic inflammation.


Microscopical findings: Treatment related findings:
Treatment related findings were observed in males and females of dose groups 3,4 and 5 (15, 50 and 150 mg/kg) of the main treatment group (experimental group A) and were still present after 4 weeks of recovery (experiment group B) in dose group 4 and 5.
The organs affected were liver, thyroid, forestomach (non-glandular stomach), small intestine, large intestine and urinary bladder.
Liver:
A liver lesion described as cholangiofibrosis was found in males and females of dose groups 3, 4 and 5 and was still present after 4 weeks if recovery in males and females of dose groups 4 and5. This, morphologically and pathogenetically very complex lesion was observed a different stages of severity and chronicity among the affected animals. A dose related increase in incidences and severity was seen in the affected dose groups.
Furthmore, males no. 25 from dose group 5, experimental group A, reacted with higher sensitivity to the test article and developed a acute course of this change leading to a mass necrosis in the liver and had to be moribund sacrificed on day 8 of the treatment period due to poor general condition. Also female no. 57 from dose group 3, experimental group A, which died during the test period on day 26 after an intestinal perforation, developed a less chronic lesion of grade 3 that was higher in severity and of more acute character than the same lesion in the other affected animals of the same dose group.
The lesion described (for technical reasons) according to the terminology of the pathology glossary used for reporting histological changes in the PatDataSystems as cholangiofibrosis, showed the same characteristics of the lesion known in the spontaneous pathology as a chronic progressive hepatitis with reflective proliferation of the intrahepatic bile duct system and beginning destruction , with partial loss, of the border cell row (regenerating hepatocytes) of the liver lobules, leading to precerrhosis of biliary type.

This lever lesion differed morphologically from the spontaneous and in rat often occurring cholangiofibrosis by producing less proliferation of the bile ducts, a distinct, active, and progressive inflammatory component , widespread fibrosis that is not limited only to the Glisson’s triangle and the tendency of developing an acute disease with mass necrosis as in male no. 35. Beside the above described cholangiofibrosis, necrosis was seen in 2/5 males (animals no. 32 and 35) of dose group 5, experimental group A, and was not found in females or in experimental group B.

Thyroid gland:
Hypertrophy of follicular epithelium was increased in dose groups 3, 4 and 5, and 5 males and 4 and 5 females of experimental group A when compared with the control group. This increase was not seen after the recovery period in experimental group B.


Forestomach:
Hperkeratosis of the cutaneous membrane of the non-glandular forestomach was found in males and females of dose groups 3, 4 and, experimental groups A, and males and females of dose groups 4 and 5, experimental group .
Additionally, ulceration was found in male no. 35 (dose group 5, experimental group A) and ulceration with foreign body granuloma (hair granuloma), and chronic inflammation in female no. 79 (dose group 5, experimental group B).


Small intestine:
Chronic inflammation was found in 2/5 males of dose group 5 and 1/5 females of dose group 3, experimental group A. In experimental group B no changes of the small intestine were seen.

In the male animals, only the duodenum was affected. In one of them (animal no. 31), the inflammation was accompanied by focal fibrosis in the mucus membrane. In the other male (animal no.35) diffuse hyperplasia of the surface epithelium was seen producing cauliflower like appearance of the cur surface of the mucus membrane.
In the affected female (n.57, a subchronic catarrhal enteritis was seen in the whole small (and large) intestine and was reported (for technical reasons) as chronic inflammation.


Small intestine:
Chronic inflammation was found in 2/5 males of dose group 5 and 1/5 females of dose group 3, experimental group A. In experimental group B no changes of the small intestine were seen.

In the male animals, only the duodenum was affected. In one of them (animal no. 31), the inflammation was accompanied by focal fibrosis in the mucus membrane. In the other male (animal no.35) diffuse hyperplasia of the surface epithelium was seen producing cauliflower like appearance of the cur surface of the mucus membrane.
In the affected female (n.57, a subchronic catarrhal enteritis was seen in the whole small (and large) intestine and was reported (for technical reasons) as chronic inflammation.


Urinary bladder:
Hyperplasia of the transitional epithelium was increased in males and females of dose groups 3, 4 and 5 of experimental group A, and in males of dose groups 4 and 5 of experimental group B.
The lesion was graded as moderate in female no. 57 (dose group 3, experimental group A), slight in female no. 73 (dose group 5, experimental group A) and minimal in all other affected animals.

Other examinations:

Findings not related directly to treatment:
Thymus and mesenteric lymph nodes diminished in size were found in 1/5 males of dose group 5, experimental group A, and were the result of a general atrophy of lymphatic tissue not directly related to the treatment. Enlarged adrenals found 1/5 males of dose group 5, experimental group A, were produced by cortical hypertrophy considered not directly related to the treatment. A few other macroscopical findings were observed in this study. They occurred in comparable numbers and were similar to those occurring spontaneously in laboratory rats of this strain and age. Thus, no experimental relevance is attributed to these findings.

Findings not related directly to treatment:
In relation to poor general condition in male no. 35 (dose group 5, experimental group A)and in emaciation in female no. 57(dose group 3, experimental group A), a general atrophy of lymphatic tissue was seen. The affected organs were thymus, splenic white pulp, mesenteric lymph node, Peyer’s patches and axillary node.
In relation to the inflammatory process in the gastrointestinal tract and abdominal cavity found in these two animals, macrophages (reported as phagocytic cells) were increased in the spleen.
Furthermore, hypertrophy and haemorrhage of the adrenal cortex was seen in both animals and was explained as a sign of general stress produced by the severe gastrointestinal and hepatic disorder leading to poor general condition and emaciation.
Additionally, some other changes were found in this study. They commonly occur in laboratory rats of this strain and age, and, neither their incidences nor their distribution or morphological appearance gave any indication of a treatment related association.

Statistics:

Statistical anlaysis of in-life and organ weight data were carried out using Statistical routines contained in the NOVATOX System.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY:
One male of the high dose groups (no. 35, 150 mg/kg) was sacrificed in moribund condition at study day 8. One female of group 3 (no. 57, 15 mg/kg) was found dead at study day 26. All other animals survived to scheduled necropsies.
5/10 high dose males (150 mg/kg) showed no clinic signs, the others showed piloerection (3/10), salivation (3/10), dyspnea (1/10), decreased muscle tone (1/10), hunched/recumbent position (1/10), aggressiveness (2/10), and paleness.

BODY WEIGHT AND WEIGHT GAIN:
Significantly depressed mean body weight development was noted for high dose males (150 mg/kg) from day 5 onwards. This resulted in a difference to the control mean of -13% at day 28 (gain -33%0. A clear normalzation towards the end of the recovery period was noted.
The mean body weight development in females was not influeced by treatment.

FOOD CONSUMPTION:
Significantly lower food intakes (g/animal/day) were recorded for high dose males (-32%) and females (-22%) during the first treatment week. This resulted in anoverall intake of -12% by high dose males, whe compared with the contro group wheras the overall female intake was comparable with the control group.
A significantly increased food consumption was noted for high dose males during the firest two weeks (+9 to 10%) of the recovery period.

WATER CONSUMPTION:
A significantly higher water intake by group 4 and 5 males during week 2 was considered an isolated and incidental finding of no toxicological relevance.

HAEMATOLOGY:
At the end of the treatment period, a slight leucocytosis was recorded for males and females of group 5 (150 mg/kg) associated with higher counts for neutrophils, monocytes and large unstained cells in males and with higher counts for all cell types (neutrophils, eosinophils, basophils, lymphocytes, monocytes and large unstained cells) in females. In addition, males of group 5 had thrombocytosis. Furthermore, prothrombin activity was increased in males and females of group 5 (150 mg/kg) and in males of group 4 (50 mg/kg). The treatment had no influence on red blood cell parameters in males, while a lower haemoglobin concentration was recorded for females of group 5 (150 mg/kg).
Following a 4-week recovery period, values of the above parameters returned to values similar to those recorded for controls, indicating reversal of the treatment effects.

Although a number of inter-group differences attained a level of statistical significance they were not related to treatment as they did not form a dose-responsive relationship and/or the magnitude of the change was too small to be of toxicological relevance. In males, these differences included a higher MCHC value for group 4 at week and a lower relative lymphocyte count for group 5 at week 5. In females, these differences included higher RDW value at week 9, a lower MCHC and a higher HDW value at week 5 for group 5, and a higher relative basophil count for group 2 at week 5.

CLINICAL CHEMISTRY:
At the end of the treatment period, males and females of group 5 (150 mg/kg) had lower values for albumin and higher values for globulin resulting in lower albumin to globulin ratios. In addition, increased activities for alanine aminotransferase and alkaline phosphates, and higher plasma cholesterol and potassium levels were recorded were recoded for males and females of group 5 (150 mg/kg). Males of the high dose group (150 mg/kg) had slightly higher plasma urea and bilirubin levels, and a higher mean value for plasma cholesterol was recorded for females for group 4 (50 mg/kg).
With the exception of bilirubin, cholesterol (males), globulin (females) and albumin to globulin ratio (males and females), all affected parameters showed full or at least partially reversibility by the end of the recovery period.
Although a few other inter-group differences attained a level of statistical significance they were not related to treatment as they did not form a dose-response relationship and/or the magnitude of the change was too small to be of toxicological relevance. In females, these differences included a lower urea value for group 5 at week 9, a lower albumin to globulin ratio for group 4 at week5, a higher triglyeride value for group 4at week 9, and a higher potassium value for group 2 at week 5.


URINALYSIS:
The treatment did not influece the urine parameters investigated.

NEUROBEHAVIOUR:
No treatment signs were observed I treated animals and all functional measurements were comparable to those of the control compound.
Group comparisons using multiple t-tests did not show statistically significant differences between treated and control group.
Motor activity measurements conducted at the end of the treatment and recovery periods did not reveal any compound-related effects in groups 2 to 5 as compared to control. Group comparisons using multiple t-tests that were corrected for multiple testing using bootstrap resampling did not reveal any difference between group 2 and 5 and controls.

ORGAN WEIGHTS:
The absolute/relative mean liver weights were affected in group 5 males (+9%n.s. / +23%) and in females of group 3 (+17 %n.s. / +16%), 4 (+11%n.s. / +11%n.s1.), and 5 (+36% / +38%). At recovery end complete reversal of liver weight was noted in both sexes.
In the absence if histological finding the changes to abs./rel. mean adrenal weights in males of groups 3 (-24% / -23%n.s.), 4 (-22% / -19n.s.), and 5 (-21% / -11%n.s.) were considered of secondary nature, probably related to stress. No relevant changes were noted at recovery end.
At recovery and additional changes were noted to abs./rel. weights of tests (-14%/-11%n.s.), ovary (-33% / -33%), spleen (+63% / +68%), heart (+8%n.s. / +12%), and kidney (+11%n.s. / +15%). However, in the absence of histological findings at both treatment and recovery end these changes were considered of secondary nature and of no toxicological relevance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the results of this study a No-Observable-Adverse-Effect Level (NOAEL) of 5 mg/kg body weight per day was defined for animals of both sexes.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test in albino Wistar rats, oral treatment with CA 2215 A at dose levels of 5, 15, 50 and 150 mg/kg by daily gavage for 28 consecutive days produced unspecific clinic signs in a few individuals and early deaths at 15 and 150 mg/kg and a number of unspecific clinical signs in a a few individuals at ≥ 15 mg/kg body weight. Body weight development was affected in high dose males only. A remarkable individual variation in susceptibility was noted.
The major target organs identified were the forestomach presenting with hyperkeratosis, the liver with cholangiofibrosis, and the urinary bladder with hyperplasia of the transitional epithelium at dose levels ≥ 15 mg/kg body weight.
A number of changes to blood chemistry parameters as well as morphological changes to forestomach and liver tissues persisted through the recovery period and showed only incomplete reversal.
Based on the results of this study a No-Observable-Adverse-Effect Level (NOAEL) of 5 mg/kg body weight per day was defined for animals of both sexes.

Executive summary:

Oral administration of CA 2215 A (Intermediate of CGA 276854) to rats by daily gavage resulted in pronounced systemic toxicity in some animals treated at dose level of 15 to 150 mg/kg as substantiated by early deaths (one male, one female), marked non-specific clinical signs in some males and females), and marked effects on body weight development (males only) and diet intake. In general, males tended to be affected more severely than females and the range of incidences and gradings of different histological lesions observed in this study point on remarkable differences in individual susceptibility and reactions towards the test item. 

The early death of male no. 35 (group 5, 150 mg/kg) and female no. 57 (group 3, 15 mg/kg) was subject of detailed investigation during which these fatalities were clearly associated with treatment. Male no. 35 (group 5) died on day eight developed a massive liver lesion with mass necrosis leading to death. Female no. 57 (group 3) died after an intestinal perforation with purulent inflammation in the abdominal and pelvic cavity following subchronic inflammatory changes in small and large intestine. Intestinal perforation in female no. 57 and almost perforation of the stomach in another female (no. 79 group 5) were further complications of the main changes originating from the irritation of the mucus membrane of the gastrointestinal tract by the test article or its metabolic products assumingly excreted over the hepatic bile system and the gut. Excretion of the test item or metabolic products thereof are considered responsible for a minimal to slight hyperplasia of the transistional urinary bladder epithelium also found at ≥ 15 mg/kg body weight at treatment end and at ≥ 50 mg/kg after recovery. 

The major pathological lesions were chronic progressive hepatitis leading to a picirrhosis (reported, for technical reasons, in the tables as cholangiofibrosis) and hyperkeratosis in the non-glandular stomach (forestomach) at ≥ 15 mg/kg at treatment end at ≥ 50 mg/kg after recovery. Ulceration of the cutaneous membrane of the forestomach and chronic inflammation in the small and large intestine were found additionally in few animals which seemed to be higher susceptible to the test item. An increased incidence of hypertrophy of the thyroid follicular epithelium found at ≥ 15 mg/kg body weight but not at recovery end, represents an adaptive syndrome following a variety of hepatic activities. Findings in the liver, forestomach, and urinary bladder were persistent throughout the recovery period. 

The neurotoxicological investigations including functional observational battery and motor activity revealed no treatment-related effects. In addition, microscopial examination of central and peripheral nervous system, eyes and muscle did not evidence any compound-related changes. Therefore, it can be excluded that the test item induced neurotoxicity in these animals. 

Hermatological parameters were affected in males at 50 mg/kg (prothrombin activity) and in both sexes at 150 mg/kg (leukoctosis) . In addition, a lower haemoglobin concentration was noted in high dose females. A complete reversal was noted in both sexes at recovery end. 

Changes to blood chemistry parameters included disturbed protein ratios, increased activity of liver enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase), and increased plasma levels for cholesterol, potassium, urea and bilirubin in high dose animals. Plasma cholesterol reversal was noted for liver enzyme activity, plasma urea and potassium levels, whereas incomplete recovery was noted for proteins, bilirubin, and cholesterol. 

 

Under the conditions of this test in albino Wistar rats, oral treatment with CA 2215 A at dose levels of 5, 15, 50 and 150 mg/kg by daily gavage for 28 consecutive days produced unspecific clinic signs in a few individuals and early deaths at 15 and 150 mg/kg and a number of unspecific clinical signs in a a few individuals at≥15 mg/kg body weight. Body weight development was affected in high dose males only. A remarkable individual variation in susceptibility was noted. 

The major target organs identified were the forestomach presenting with hyperkeratosis, the liver with cholangiofibrosis, and the urinary bladder with hyperplasia of the transitional epithelium at dose levels≥15 mg/kg body weight. 

A number of changes to blood chemistry parameters as well as morphological changes to forestomach and liver tissues persisted through the recovery period and showed only incomplete reversal.

Based on the results of this study a No-Observable-Adverse-Effect Level (NOAEL) of 5 mg/kg body weight per day was defined for animals of both sexes.