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Description of key information

NOAEL for a 28-day repeated dose study of a chemical category member, TSA, was 50 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP
Qualifier:
according to guideline
Guideline:
other: OECD 421
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3500
Principles of method if other than guideline:
28-day repeated dose with reproductive toxicity screening study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System:
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within ± 20% of the mean weight.
The range of the body weight was:
Females: 173-200g, (mean: 189.18 ± 20%= 37.84 g)
Males: 290-325g, (mean: 303.83 g, ± 20%= 60.77 g)

Number of animals: 10 Males and 10 females per group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

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.0906)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.030512)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 4 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method for TSA was received from the sponsor and modified for this study. The method was validated for a concentration range of 10-70 mg/ml in corn oil. Recoveries of all the test samples were between 46 and 153% of nominal. A few samples were analysed a second time to investigate possible errors, resulting in recoveries for all samples as between 78 and 153% of nominal. Analysis was by gas chromatography (GC). Parameters and equipment:
GC: Agilent 6890
Columns: DB-5 MS (30 m*0.25 mm, 0.25 µm film thickness), HP-5 (30 m*0.25 mm, 0.25 µm film thickness)
Carrier gas: Helium 5.0
Split: 20:1
Initial Gas Flow: 2.0 mL/min
Injector Temperature: 250 degrees C.
Injection volume: 1 µl
Column Oven: 40 degrees C (2 min), ramp: 30 degrees C per min to 290 degrees C (3 min).
Detector: MS
Run time: 13.33 min. The test item eluted as a set of peaks at approximately 7.5 min and 7.9 minutes.
Source Temp: 230 degrees C
Ions monitored: 109.2, 124.3 and 195.2 amu
Documentation: exact chromatographic conditions are documented in the raw data and final report.

Stock solution: 100.13 mg of test item was weighed precisely into a 100 ml volumetric flask and filled with acetone to obtain a clear colourless solution.
Standards: Appropriate amount of the stock solution were diluted with acetone to obtain 6 calibration standards ranging from 50 to 500 mg test item/L.
Fortified samples were prepared in a similar manner and diluted with corn oil instead of acetone. Five replicates and two blanks were prepared and analysed.

Sample preparation: Specimens were taken out of the freezer and allowed to thaw at room temperature and the total weight was recorded. The specimens were homogenised by vortexing. Each was transferred entirely into a volumetric flask (50 ml) and the volume brought to the mark with acetone. It was observed that after a while in some of the samples small amounts of precipitate had formed. The sample solutions were further diluted with acetone and then analysed. Each specimen was analysed at least once. Six specimens were analysed two or three times.

Quantification: The external standard method was used. Measured samples were quantified by measuring the peak area with reference to the calibration curve of the standard solutions.

LOD: The Limit of Detection (LOD) of the method is defined as the lowest concentration having a peak height equivalent to or better than three times the baseline noise.
LOQ: The Limit of Quantification (LOQ) of the method was determined as the lowest fortification level at which acceptable recovery (70 to 110% of nominal) was obtained.

Regression Coefficient: at least 0.09988
Detection Limit: 1.59 mg test item/L.
Quantitation limit: 10 mg test item/ml after dilution by a factor of approximately 100.
Repeatability: Two standard solutions were injected 5 times and the response factor was calculated. The relative standard deviation was 1.7% and 1.2 %, respectively.
Mean recovery rate: at 10 mg/ml: 93 % of nominal (n=8; SD=12%).
at 35 mg/ml: 95 % of nominal (n=9; SD=2%).
at 70 mg/ml: 95 % of nominal (n=5; SD=2%).


Duration of treatment / exposure:
28 days (males), 54 days (females)
Frequency of treatment:
daily, 7 days per week
Remarks:
Doses / Concentrations:
50, 150 and 250 mg/kg bw/d
Basis:
other: nominal ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 50, 150, 250 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C)

The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 4 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
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 the females were checked to confirm the evidence of mating. 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.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period and post mating period in males.

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. 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 and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28 days) and females were sacrificed on respective post natal day 4 along with pups by using high dose of Ketamine/Xylazine (2:1). At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.

The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Additional organs of animals which died during the course of the study were preserved and examined histopathologically in order to determine the cause of death.


Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female and kidneys from few adult females were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken.


Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.
Observations and examinations performed and frequency:
Parental animals: Observations and examinations
Males and Females: Body Weight, Food Consumption, Clinical Signs, Mating behaviour, organ weight, Gross pathology and Histopathology.
Sacrifice and pathology:
Postmortem examinations (Parental animals)
Males- Day 29; Females: On PND 4
Other examinations:
Estrous cyclicity (Parental animals)
Not evaluated
Sperm parameters (Parental animals)
Not evaluated
Litter observations
Number and sex of pups, still births, runts, gross abnormalities of the pups and litter weight
Reproductive indices
Copulation Index (%), Fertility Index (%) and Delivery Index (%)
Offspring viability indices
Viability Index (%)

Statistics:
A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
not examined
Details on results:
Mortality:
One male animal (21) from the MD group was found dead on premating day 6.
In females, 8 animals (71, 72, 73, 74, 75, 77, 78 and 80) from HD group died on premating day 5, 5, 6, gestation day 11, 21, 22, 23 and 21, respectively.

Clinical Observation:
Test item related clinical signs were observed in male and female MD and HD group animals. Predominant clinical signs observed in male animals were slight salivation (3/10 in MD and 9/10 in HD), moderate salivation (2/10 in HD), slight piloerection (4/10 in MD and 6/10 in HD), moderate piloerection (2/10 in MD and 1/10 in HD), moving the bedding (7/10 in MD and 10/10 in HD) and abnormal breathing (1/10 in HD).

In females, predominant clinical signs observed were slight salivation (2/10 in LD, 6/10 in MD and HD), moderate salivation (4/10 in MD and HD), slight piloerection (1/10 in C, 6/10 in MD and HD), moderate piloerection (1/10 in MD and 3/10 in HD), moving the bedding (9/10 in MD and 6/10 in HD) and clonic convulsion and tremor (1/10 in HD).


Body Weight and Body Weight Change:
In males, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, biologically significant decrease in body weight and bodyweight change was observed in MD and HD group when compared with controls.

In females, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups. However, a biologically significant decrease in body weight and bodyweight change was observed from gestation day 20 to postnatal day 4 in MD and HD group when compared with controls.


Food Consumption:
In males, statistically significant decrease in food consumption during premating day 1-7 and biologically significant decrease during premating day 7-14 was observed in MD and HD group when compared with controls. This effect on male food consumption could be considered as treatment related.

In females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males. The few spontaneous gross pathological findings observed in male animals were yellow spot on right epididymides (1/10 in MD males) and yellow spot on left epididymides (1/10 in HD males).

In females, predominant gross pathological findings observed in dead animals were white area on left liver lobe (1/10 in HD females), discoloured stomach (3/10 in HD females), gaseous distension of intestinal tract (3/10 in HD females), dark spot on lung (1/10 in HD females), discoloured red thymus (2/10 in HD females) and small thymus (1/10 in HD females)

These gross pathological findings observed in males were spontaneous in nature. However, the significance of these macroscopic findings in dead females remains unclear and an influence of beginning of autolysis cannot be excluded.

Organ Weight:
In males, statistical analysis of organ weight data revealed a significant decrease in absolute prostate and seminal vesicles with coagulating gland weights in HD group when compared with controls.

In females, a statistically significant increase in relative kidney weights (left and total) was observed in MD and HD group when compared with controls.

Since in males, decrease in absolute prostate and seminal vesicles with coagulating gland weights was minimal and no effect on relative weight was observed, this effect on male weights was considered to be non adverse. However, in the light of histopathological findings in the kidneys, effect on kidney weights in females was considered to be test item related.

Histopathology
One male rat from the MD group and eight females from the HD group were found dead during the treatment period. Gavage accident was established as direct cause of death for one decedent high dose female. In addition, in all decedents combinations of histopathological findings were noted in several of the following organs: kidney, forestomach, lymphoid organs (spleen, thymus, Peyer's patch, mesenteric and axillary lymph node), lung, adrenal gland and heart, and were considered to have contributed to the death of these animals.

Renal tubular degeneration/regeneration was considered to be possibly directly test item-related. In the forestomach, minor (sub)mucosal mixed cell infiltration, submucosal congestion/hemorrhage and (peri)vasculitis were also considered to represent direct effects of the test item. For lymphoid atrophy and single cell death of lymphoid organs, as well as for leukocytostasis and vasculitis/thrombi in the lung, a direct test item relationship was not clear. Adrenal gland changes (cortical degeneration/necrosis and diffuse cortical hypertrophy) and heart changes (cardiomyocyte vacuolation, focal myocardial degeneration) were considered to be secondary.

At terminal sacrifice, the kidney was evaluated in all surviving females. Against a background incidence noted in control females, minimal to moderate numbers of basophilic (regenerative) tubules in the cortex and medulla occurred in the females of all three dose groups, without a clear dose relationship. Renal changes were considered to be possibly directly test item-related.

Altogether, there was no evidence of a direct test item-related effect on male or female reproductive organs in this study.



Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: signs of toxicity in both male and females (primarily females) and mortalities in 250 mg/kg bw/d group females and 150 mg/kg bw/d group males.
Critical effects observed:
not specified
Conclusions:
Repeated dose administration of a category member, Tripropenyl Succinic Anhydride, to groups of 10 male (28 days) and female (maximum 54 days) Wistar rats at dosages of 50, 150 and 250 mg/kg body weight revealed mortality (primarily among pregnant females), but no significant findings of toxicological relevance in HD group females. Males displayed a decrease in food consumption and decreased body weight gain in the MD and HD groups. Based on the data generated from this reproduction/ developmental toxicity screening test with Tripropenyl Succinic anhydride, the no observed adverse effect level (NOAEL) was considered to be 50 mg/kg body weight/d. Data can be read-across among members of the C8-12 Alkenyl Succinic Anhydrides Category, based on common functional groups, similar break-down products and potency patterns among carbon-chain length. This is adequate to fulfill the information requirements, to be the basis for classification and labelling decisions, and for risk assessment.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Adequate

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A chemical category member, tripropenyl succinic anhydride (TSA) was tested in a 28-day repeated dose toxicity study (OECD 421) in Wistar rats, at doses of 50, 150 and 250 mg/kg bw/day, in a corn oil vehicle. The NOAEL was 50 mg/kg bw/d. There is also a 14 -day dose range finding study on the registered substance, tetrapropenyl succinic anhydride (TPSA). No significant toxicity was found at doses up to 300 mg/kg bw/d. The WHO reviewed the human health risks of cyclic acid anhydrides, and, while data are limited, did not find a weight of evidence which suggests that repeated dose exposure confers a toxicity risk. The human health risks which were identified pertain to the immediate reactivity of the anhydride group, which can manifest as irritation and sensitisation.

Risk management measures are proposed to minimize acute exposure to the anhydride.  It is proposed that, if any additional testing is required to be conducted, it be performed on the more water-soluble cleavage product of the anhydride, as all the anhydrides are hydrolytically labile.

A chemical category is established for alkenyl succinic anhydrides with C8-C12 alkenyl side chains, based on common functional groups, similar physico-chemical properties, common breakdown/metabolic products via physical and biological processes, and a constant pattern in the changing of the potency across the category.  These include tetrapropenyl succinic anhydride (CAS 26544-38-7), octenyl succinic anhydride (CAS 26680-54-6), n-dodecenyl succinic anhydride (CAS 19780-11-1) and tripropenyl succinic anhydride (CAS 28928-97-4). Common functional groups are a dihydro-2,5 -furandione (cyclic anhydride) ring, a carbon chain of length 8 to 12 carbons (with or without branching methyl groups), and one double bond in the carbon chain, location unspecified.  Common breakdown products are the dioic acids of the corresponding anhydride. A constant pattern may also be displayed in acute toxicity, dermal irritancy and biodegradation, with the lowest carbon chain length (C8) displaying the highest irritation potential in vivo and highest biodegradation potential. Irritation and degradation diminishes with increasing carbon chain length. Read-across is appropriate to fill the data gaps for repeated dose toxicity.

Justification for classification or non-classification

No findings from repeated dose toxicity testing of a category member, tripropenyl succinic anhydride, meet criteria for classifcation of specific target organ toxicity-repeated exposure. There is currently insufficient evidence to require classification according to Regulation EC No. 1272/2008.