Amines, rosin

Endpoint summary

Description of key information

In a key combined repeated dose, reproductive/developmental toxicity study, Gum Rosin was administered to rats (12/sex/dose) in dietary mixtures at concentrations of 0, 2500, 5000, and 10000 ppm for a period of 50 days for male rats and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (at least 36 days, up to 53 days).

The treatment with the test item did not lead to any premature mortalities and had no effects on behaviour, reflexes, grip strength, locomotor activity, and haematology parameters. 

In the high dose group (10000 ppm), in both sexes, significantly lower food consumption was recorded during the first treatment week. From treatment week 2 until the end of the treatment period the food consumption was slightly decreased in males, but still significantly decreased in females when compared to the control group. The lower food consumption led to a transient lower body weight gain and lower body weights until the end of the study in both sexes. Except for ruffled fur in one female from the last day of gestation to the end of the lactation period, no clinical signs were observed. The water consumption was significantly reduced during the lactation period which was possibly due to the lower number of pups and therefore lower milk production in this group.

Increased activity of alkaline phosphatase in males and increased bilirubin concentration in both sexes did not correlate with other findings and were therefore not considered adverse. Decreased absolute and relative thymus weights in females recorded at necropsy correlated with decreased lymphocytes in the cortex of the thymus. This finding was considered to be stress-related and likely not a direct effect of the test item.

In the intermediate dose group (5000 ppm), lower food consumption was recorded in males and females during the first treatment week. From treatment week 2 onwards the food consumption was similar to the control group in males, but still decreased in females. The lower food consumption led to a transient lower body weight gain in males during the pre-paring period. However, decreased body weights compared to the concurrent controls were recorded during the entire treatment period in females. No clinical signs were observed at this dose level. The water consumption of females was slightly reduced during the lactation period.

In the low dose group (2500 ppm), a transiently slightly lower body weight gain was recorded during the pre-pairing period in males. Afterwards the body weight development at this dose level was similar to the controls.

The creatinine concentration was increased at all dose levels in males. An increase of creatinine is usually a sign for kidney damage. However, no histopathological findings were recorded in the kidneys. Therefore, the increase of the creatinine was not considered to be an adverse effect. Minimal hypertrophy/vacuolation of the zona glomerulosa was observed in the adrenal glands of males from the 2500 ppm group and in females from the 5000 ppm group with dose related-incidence. The pathogenesis of this change is uncertain. The zona glomerulosa is the site of synthesis of aldosterone which is mainly involved in the control of salt and water balance in the body. Secretion of aldosterone is controlled through the renin-angiotensin system and hypertrophy of the zona glomerulosa is generally considered to be an adaptative process following stimulation of this system. Therefore, this change was not considered to be an adverse effect.

Based on the results of this study, a NOEL (No Observed Effect Level) for general toxicity could not be established due to effects on body weights at all dose levels in females during the lactation period. The NOAEL (No Observed Adverse Effect Level) for general toxicity was established at the dose level of 2500 ppm. For males, the lowest mean achieved dose level of 107.7 mg/kg/bw was derived at concertation 2500 ppm.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-22 to 2014-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The deviation did not impact the scientific validity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KB13G178
- Expiration date of the lot/batch: 2014-06-29
- Purity test date: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Bulk sample was stored frozen (-20 ± 5 °C) under nitrogen in the dark. For temperature adjustment, working samples were kept under nitrogen at room temperature (20 ± 3 °C) for a maximum of 3 hours, prior to feed preparation.
- Stability under test conditions: At least 8 days if stored at room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Species:

rat

Strain:

other: RccHan TM : WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 304 - 353 g; Females: 200 - 236 g
- Fasting period before study: No
- Housing: During acclimatization and pre-pairing in groups of up to three animals by sexin Makrolon type-4 cages, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During pairing females were housed with males (1:1) in Makrolon type-3. Following pairing period males were re-housed in their original prepairing cages and females were individually housed in Makrolon type-3 cages. All cages were provided with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands). In addition a wood stick was provided (batch nos. 02105130819, 77, 6960C-100267, 6960C-101212, 122207 and 132211).
- Diet (e.g. ad libitum): Microgranulated standard Harlan Teklad 2018C (batch no. 41/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitumin water bottles.
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
IN-LIFE DATES: From: 2013-10-22 To: 2013-12-22

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared at intervals not exceeding 8 days using the test item as supplied by the supplier.
- Mixing appropriate amounts with (Type of food): Gum Rosin, CAS no. 8050-09-7, was ground to powder using an electrical grinder, weighed into a tared glass beaker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. No heat was used during diet preparation. Control feed for the animals of group 1 was prepared similarly, but without test item.
- Storage temperature of food: Stability of the test item in feedwas at least 8 days according to the results of stability analyses performed within the Harlan Laboratories study no. D80871 (Method Implementation and Development; non-GLP). Feed preparations were stored at room temperature protected from light in metal containers until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period and at the end of gestation/start of lactation period (i.e. two occasions). Stability of the test item in the feed for 8 and 28 days at room temperature was determined at the start of the pre-pairing period (i.e. one occasion). For assessment of content and homogeneity, a 100 g sample was collected from each of the top, middle and bottom of every dietary admixture ofthe respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation. For assessment of stability, a 100 g sample was drawn from the middle of the dietary admixture on the day of preparation and stored at roomtemperature for 8 or 28 days. After preparation the samples were delivered to the analytical department at ambient temperature and stored there frozen (at -20 ± 5 °C) until analysis. The samples were analyzed by HPLC coupled toan ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was usedas the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were discarded after finalization of the report without any further note.

Duration of treatment / exposure:

Males: 50 days (during 14 days pre-pairing period, and 36 days pairing period and postpairing period).
Females: Minimum 5 weeks (14 days pre-pairing period, up to 17 days pairing period, approximately 21 days of gestation period and lactation period to day 4 post partum).

Frequency of treatment:

Continuously (ad libitum)

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

2 500 ppm

Remarks:

Low Concentration

Dose / conc.:

5 000 ppm

Remarks:

Intermediate Concentration

Dose / conc.:

10 000 ppm

Remarks:

High Concentration

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were selectedbased on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study no. D80882, using concentrations in feed of 5000, 10000 and 20000 ppm and resulting in adverse reduction of food consumption, body weight loss and early termination of males and females treated with feed containing test item at a concentration of 20000 ppm.A dietary inclusion level of 10000 ppm resulted in approximately 6% (males) and 10% (females) reduction in body weight compared to the control group during 15 days of treatment.
- Rationale for animal assignment (if not random): Performed after at least three days of acclimatization using a computer-generatedrandom algorithm. Bodyweights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: Once prior to the first administration of the test item and weekly thereafter.
Females: Once prior to the first administration of the test item, weekly during the pre-pairing and pairing and on days 0, 6, 13 and 20 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Once during acclimatization, at three-day intervals during pre-pairing and postpairing periods and weekly during pairing period.
Females: Once during acclimatization, at three-day intervals during pre-pairing period, weekly during pairing period and on the following days: 0, 3, 6, 9, 12, 15, 18, 21 post coitum and 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Males: At three-day intervals during pre-pairing and post-pairing.
Females: At three-day intervals during pre-pairing and gestation, and from days 1 to day 4 post partum in all females (except nos. 50, 53, 56, 58, 62, 65, 69, 74, 77, 79, 80, 82, 85, and 89 for which no food consumption was recorded during the lactation period due to a planning error). No food consumption was recorded during the pairing period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION INTAKE: Yes
- Time schedule for examinations: Daily water intake was measured gravimetrically during pre-pairing, post-pairing, gestation and lactation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All parent animals were examined macroscopically for any structural changes at the scheduled necropsy. Special attention was directed at the organs of the reproductive system. The uteri of all dams were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites and the number of implantation sites were noted. The number of corpora luteain each ovary was recorded for all pregnant females.

HISTOPATHOLOGY: Yes The tissues indicated in Table 2 and Table 3. were prepared for microscopic examination and weighed, respectively.

Parameters examined in male parental generations: [other: spermatogenesis]

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, food and water consumption, body weights, clinical laboratory investigations, organ weights, macroscopical findings, and reproduction data:
• Means and standard deviationsof various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
• Kruskal Wallis for the righting reflex.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ruffled fur was recorded in one female of group 4 from the last day of gestation to the end of the lactation period. This was considered to be related to the treatment with the test item. No clinical signs were observed in females of groups 2 and 3 and in males at any dose level.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males - A dose-related effect of the test item on body weight development was recorded at all dose levels. During the pre-pairing period lower body weight gain was recordedin all test item-treated groups (6.2%, 4.9%, and 0.6% in groups 2, 3, and 4 on day 7) when compared to the control group (8.6%). During the post-pairing period the body weight gainwas similar in all groups. Weights were decreased in males of group 4 fromtreatment day 4 until the end of the study but remained within 10% of concurrent control group values. No significant effects on body weights were recorded in males of groups 2 and 3.

Females - A dose-related effect of the test item on body weight development was recorded at all dose levels. During the pre-pairing period lower body weight gainwas recorded in females of groups 3 and 4 (0.2%, and -3.4% on day 7) when compared to the control group (5.1%).During the gestation period the body weight gain in females of group 3 and 4 was still lower (46.9% and 37.6% vs. 56.7% in the control group on day 21). During the lactation periodno significant differences in
body weight gain were recorded. Weights were decreased in females of groups 3 and 4 from treatment day 4 until the end of the study. Body weights in females of groups 3 and 4 were within 10% of the concurrent control value during the pre-pairing period. In group 3, body weights remained at 87-93% of control for the remainder of the study. In group 4, the mean body weight fell to about 80% of the concurrent control value at the end of the gestation period and throughout the lactation period. In group 2, slightly lower body weights wererecorded during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males - Test item-related effects on food consumption were recorded in group 3 and 4. Lower food consumption (-42%) was recorded in males of group 4 during the first treatment week when compared to the control group. From treatment week 2 until the end of the treatment period the food consumption was slightly decreased (-12% during the second week of the prepairing period and -8% during post-pairing period). Slightly lower food consumption (-15%) was recorded in males of group 3 during the first treatment week. From treatment week 2 onwards the food consumption was similar to the control group. No effects were recorded in males of group 2.

Females - Test item-related effects on food consumption were recorded in group 3 and 4. Lower food consumption (-53%) was recorded in females of group 4 during the first treatment week when compared to the control group. From treatment week 2 until the end of the treatment period the food consumption was still decreased (-20% during gestation and -45% during lactation). Lower food consumption was recorded in females of group 3 during the whole treatment period (-16% during pre-paring, -14% during gestation and -37% during lactation) when compared to the control group. No effects were recorded in females of group 2.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males - A dose-related effect of the test item on food conversion efficiency was recorded at all dose levels. Lower food conversion efficiency (-55%) was recorded in males of group 4 during the prepairing period when compared to the control group. During post paring the food conversion efficiency was similar to the controls. Lower food conversion efficiency was recorded in males of groups 2 (-25%) and 3 (-31%) during the first four days of treatment when compared to the control group. Afterwards the food conversion efficiency was similar to the controls.

Females - A dose-related effect of the test item on food conversion efficiency was recorded at all dose levels. Lower food conversion efficiency was recorded in females of groups 3 and 4 during the first four days of the pre-pairing period when compared to the controls. Afterwards the food conversion efficiency was similar to the control group. At the end of the gestation lower food conversion efficiency was recorded in all test item-treated groups. During the lactation period the feed consumption in all test item-treated groups was similar to the control group.

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

effects observed, treatment-related

Description (incidence and severity):

Males - No effects of the treatment with Gum Rosin on water consumption were recorded.

Females - During the lactation period the water consumption was decreased by 33% in females of group 4 and by 20% in females of group 3. This was considered to be a test item-related effect. No effects of the treatment on water consumption were in recorded in females during the prepairing and gestation period. No effects were recorded in group 2.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects of the treatment with Gum Rosin on hematology parameters were recorded. Changes in a few parameters achieving individual statistical significance were considered to be unrelated to treatment, because they lacked a dose-relationship and/or were clearly within the historical control range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males - The following changes were considered to be test item-related:
• The activity of alkaline phosphatase was increased in males of group 3 by 48% and by 61% in group 4.
• The total bilirubin concentration was increased in males of group 4 (8.40 µmol/L vs. 0.43 in the controls).
• The creatinine concentration was increased at all dose levels (+19%, +32%, and +47% in groups 2, 3, and 4, respectively).

Increased triglyceride concentration in group 2 reaching statistical significance was considered to be incidental, because the values were within the range of the historical controls and this change was not dose-related.

Females - Increased concentration of total bilirubin in females of group 4 (3.27 µmol/L vs. 0.00 in the controls) was considered to be treatment-related. Changes in a few other parameters reaching statistical significance were considered to be incidental, because the values were within the range of the historical controls and/or the changes were not dose-related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

Grip Strength: No effects of the treatment with Gum Rosin on grip strength (fore- and hind paws) were recorded.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decreased relative and absolute thymus weights recorded in females of group 4 were considered test item-related. Decreased absolute heart, thyroid and prostate weights, increased relative (to body weight) weights of brain and liver, as well as decreased relative (to brain weight) weights of heart, thyroids, prostate and kidneys in males of group 4 were consideredto be either effects caused by the lower body weights in this group or incidental findings, because they were either not dose related and/or no correlating histopathology findings were recorded. Decreased absolute kidney, and adrenal weights offemales at all dose levels, decreased absolute ovary and heart weights, and increased relative (to body weight) brain weights of group 3 and 4 females, increased relative (to body weight) weights of kidneys in group 4 females, decreased absolute pituitary and liver weights in group 4 females, as well as decreased weights relative to the brain weight of pituitary glands, liver, and spleen in group 4 females, decreased weights relative to the brain weight of heart, kidneys, adrenals and ovaries in group 3 and 4 females, decreased absolute thymus weights in group 2 and 3 females, and decreased absolute spleen weights in group 3 and 4 females were considered to be either effects caused by the lower body weights in this group or incidental findings, because they were either not dose-related and/or no correlating histopathology findings were recorded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were recorded.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In adrenals, minimal hypertrophy/vacuolation of the zona glomerulosa was recorded in 0/12, 2/12, 5/12 and 9/12 males of groups 1, 2, 3, and 4, respectively and 0/12, 0/12, 3/12 and 5/12 females of groups 1, 2, 3, and 4, respectively. The change was characterized by diffuse hypertrophy of the zona glomerulosa associated with vacuolization of the zona glomerulosa cells. Decreased lymphocytes in the cortex of the thymus was observed in 0/12, 0/12, 0/12 and 6/12 females of groups 1, 2, 3, and 4, respectively, changes being minimal, slight, moderate and marked in 3/5, 1/5, 1/5 and 1/5 females respectively. Of note, moderate focal erosion/ulcer was observed in the non-glandular gastric mucosa of rat no. 93 (group 4).

Other effects:

no effects observed

Description (incidence and severity):

Locomotor Activity: No effects of the treatment with Gum Rosin on locomotor activity were recorded.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic Toxicity

Effect level:

ca. 2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic Toxicity

Effect level:

ca. 107.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: The lowest mean achvieved dose level corresponding to 2500 ppm (post-pairing period)

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

other: Body weight and body weight gain

Organ:

other: Body weight

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 4. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PRE-PAIRING PERIOD
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	325	328	330	328
	ST. Dev	10.3	7.1	9.2	12.9
	N	12	12	12	12
Day 4	Mean	344	342	341	326**
	ST. Dev	10.3	7.5	9.6	10.9
	N	12	12	12	12
Day 7	Mean	353	349	346	330**
	ST. Dev	11.7	8.7	10.3	9.1
	N	12	12	12	12
Day 10	Mean	361	354	352	332**
	ST. Dev	13.0	10.7	10.4	11.3
	N	12	12	12	12
Day 13	Mean	369	367	362	342**
	ST. Dev	12.9	11.7	11.6	13.3
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PAIRING
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 2	Mean	373	367	363	338**
	ST. Dev	15.0	12.6	11.0	16.4
	N	12	12	12	12
Day 8	Mean	387	381	378	348**
	ST. Dev	15.1	13.6	14.2	18.3
	N	12	12	12	12
Day 15	Mean	400	395	393	361**
	ST. Dev	18.9	16.7	16.3	15.6
	N	12	12	12	12

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - POST-PAIRING
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	416	404	404	375**
	ST. Dev	18.6	16.8	17.5	15.5
	N	12	12	12	12
Day 4	Mean	419	411	404	379**
	ST. Dev	18.9	18.8	18.1	17.6
	N	12	12	12	12
Day 7	Mean	423	416	412	378**
	ST. Dev	18.0	19.2	16.5	17.8
	N	12	12	12	12
Day 10	Mean	425	421	415	382**
	ST. Dev	19.7	21.2	18.0	16.3
	N	12	12	12	12
Day 13	Mean	425	423	418	385**
	ST. Dev	19.2	22.9	18.1	16.0
	N	12	12	12	12
Day 16	Mean	428	425	419	383**
	ST. Dev	18.9	23.9	18.3	17.7
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - PRE-PAIRING PERIOD
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	219	220	217	216
	ST. Dev	9.3	8.6	8.4	8.0
	N	12	12	12	12
Day 4	Mean	227	227	215**	206**
	ST. Dev	7.3	13.2	8.1	9.2
	N	12	12	12	12
Day 7	Mean	230	231	217*	209**
	ST. Dev	8.4	13.9	9.1	10.9
	N	12	12	12	12
Day 10	Mean	235	232	222*	211**
	ST. Dev	10.0	13.8	9.1	10.3
	N	12	12	12	12
Day 13	Mean	237	236	225*	213**
	ST. Dev	11.7	15.2	7.3	12.1
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 8. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - GESTATION
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 0	Mean	245	242	229**	220**
	ST. Dev	11.0	12.7	9.6	9.8
	N	12	10	11	11
Day 3	Mean	262	257	243**	230**
	ST. Dev	10.6	13.7	11.7	11.0
	N	12	10	11	11
Day 6	Mean	274	267	252**	239**
	ST. Dev	11.2	14.0	13.6	9.9
	N	12	10	11	11
Day 9	Mean	283	277	260**	247**
	ST. Dev	10.9	13.2	13.9	11.4
	N	12	10	11	11
Day 12	Mean	297	292	272**	256**
	ST. Dev	14.1	13.0	17.9	10.3
	N	12	10	11	11
Day 15	Mean	314	306	287**	270**
	ST. Dev	14.9	15.3	17.0	13.7
	N	12	10	11	11
Day 18	Mean	347	343	315**	291**
	ST. Dev	21.7	15.0	19.7	13.7
	N	12	10	11	11
Day 21	Mean	385	368	336**	302**
	ST. Dev	28.6	20.5	23.4	15.8
	N	12	10	11	11

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 9. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - LACTATION
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	282	263*	250**	232**
	ST. Dev	20.5	15.5	20.1	12.3
	N	11	11	11	11
Day 4	Mean	296	280*	257**	237**
	ST. Dev	18.0	18.3	15.1	11.8
	N	12	11	11	11

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. Test item intake of Males during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	151.5
P	5000	285.2
P	10000	472.9

 

Table 11. Test item intake of Males during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0
P	2500	107.7
P	5000	209.9
P	10000	447.0

 

Table 12. Test item intake of Males during post-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	107.7
P	5000	209.9
P	10000	447.0

 

Table 13. Test item intake of Females during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	174.9
P	5000	313.7
P	10000	522.6

 

 Table 14. Test item intake of Females during Gestation Period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	183.4
P	5000	349.2
P	10000	693.2

 

 Table 15. Test item intake of Females during lactation period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	231.0
P	5000	377.6
P	10000	691.9

 

Conclusions:

Based on the results of this study, a NOEL (No Observed Effect Level) for general toxicity could not be established due to effects on body weights at all dose levels in females during the lactation period. The NOAEL (No Observed Adverse Effect Level) for general toxicity was established at the dose level of 2500 ppm. For males, the lowest mean achived dose level of 107.7 mg/kg/bw was derived at concetration 2500 ppm.

Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity study, Gum Rosin was administered to rats (12/sex/dose) in dietary mixtures at concentrations of 0, 2500, 5000, and 10000 ppm for a period of 50 days for male rats and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (at least 36 days, up to 53 days).

The treatment with the test item did not lead to any premature mortalities and had no effects on behaviour, reflexes, grip strength, locomotor activity, and haematology parameters. 

In the high dose group (10000 ppm), in both sexes, significantly lower food consumption was recorded during the first treatment week. From treatment week 2 until the end of the treatment period the food consumption was slightly decreased in males, but still significantly decreased in females when compared to the control group. The lower food consumption led to a transient lower body weight gain and lower body weights until the end of the study in both sexes. Except for ruffled fur in one female from the last day of gestation to the end of the lactation period, no clinical signs were observed. The water consumption was significantly reduced during the lactation period which was possibly due to the lower number of pups and therefore lower milk production in this group.

Increased activity of alkaline phosphatase in males and increased bilirubin concentration in both sexes did not correlate with other findings and were therefore not considered adverse. Decreased absolute and relative thymus weights in females recorded at necropsy correlated with decreased lymphocytes in the cortex of the thymus. This finding was considered to be stress-related and likely not a direct effect of the test item.

In the intermediate dose group (5000 ppm), lower food consumption was recorded in males and females during the first treatment week. From treatment week 2 onwards the food consumption was similar to the control group in males, but still decreased in females. The lower food consumption led to a transient lower body weight gain in males during the pre-paring period. However, decreased body weights compared to the concurrent controls were recorded during the entire treatment period in females. No clinical signs were observed at this dose level. The water consumption of females was slightly reduced during the lactation period.

In the low dose group (2500 ppm), a transiently slightly lower body weight gain was recorded during the pre-pairing period in males. Afterwards the body weight development at this dose level was similar to the controls.

The creatinine concentration was increased at all dose levels in males. An increase of creatinine is usually a sign for kidney damage. However, no histopathological findings were recorded in the kidneys. Therefore, the increase of the creatinine was not considered to be an adverse effect. Minimal hypertrophy/vacuolation of the zona glomerulosa was observed in the adrenal glands of males from the 2500 ppm group and in females from the 5000 ppm group with dose related-incidence. The pathogenesis of this change is uncertain. The zona glomerulosa is the site of synthesis of aldosterone which is mainly involved in the control of salt and water balance in the body. Secretion of aldosterone is controlled through the renin-angiotensin system and hypertrophy of the zona glomerulosa is generally considered to be an adaptative process following stimulation of this system. Therefore, this change was not considered to be an adverse effect.

Based on the results of this study, a NOEL (No Observed Effect Level) for general toxicity could not be established due to effects on body weights at all dose levels in females during the lactation period. The NOAEL (No Observed Adverse Effect Level) for general toxicity was established at the dose level of 2500 ppm. For males, the lowest mean achieved dose level of 107.7 mg/kg/bw was derived at concertation 2500 ppm.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2013-10-22 to 2014-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The deviation did not impact the scientific validity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KB13G178
- Expiration date of the lot/batch: 2014-06-29
- Purity test date: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Bulk sample was stored frozen (-20 ± 5 °C) under nitrogen in the dark. For temperature adjustment, working samples were kept under nitrogen at room temperature (20 ± 3 °C) for a maximum of 3 hours, prior to feed preparation.
- Stability under test conditions: At least 8 days if stored at room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Species:

rat

Strain:

other: RccHan TM : WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 304 - 353 g; Females: 200 - 236 g
- Fasting period before study: No
- Housing: During acclimatization and pre-pairing in groups of up to three animals by sexin Makrolon type-4 cages, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During pairing females were housed with males (1:1) in Makrolon type-3. Following pairing period males were re-housed in their original prepairing cages and females were individually housed in Makrolon type-3 cages. All cages were provided with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands). In addition a wood stick was provided (batch nos. 02105130819, 77, 6960C-100267, 6960C-101212, 122207 and 132211).
- Diet (e.g. ad libitum): Microgranulated standard Harlan Teklad 2018C (batch no. 41/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitumin water bottles.
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
IN-LIFE DATES: From: 2013-10-22 To: 2013-12-22

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared at intervals not exceeding 8 days using the test item as supplied by the supplier.
- Mixing appropriate amounts with (Type of food): Gum Rosin, CAS no. 8050-09-7, was ground to powder using an electrical grinder, weighed into a tared glass beaker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. No heat was used during diet preparation. Control feed for the animals of group 1 was prepared similarly, but without test item.
- Storage temperature of food: Stability of the test item in feedwas at least 8 days according to the results of stability analyses performed within the Harlan Laboratories study no. D80871 (Method Implementation and Development; non-GLP). Feed preparations were stored at room temperature protected from light in metal containers until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period and at the end of gestation/start of lactation period (i.e. two occasions). Stability of the test item in the feed for 8 and 28 days at room temperature was determined at the start of the pre-pairing period (i.e. one occasion). For assessment of content and homogeneity, a 100 g sample was collected from each of the top, middle and bottom of every dietary admixture ofthe respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation. For assessment of stability, a 100 g sample was drawn from the middle of the dietary admixture on the day of preparation and stored at roomtemperature for 8 or 28 days. After preparation the samples were delivered to the analytical department at ambient temperature and stored there frozen (at -20 ± 5 °C) until analysis. The samples were analyzed by HPLC coupled toan ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was usedas the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were discarded after finalization of the report without any further note.

Duration of treatment / exposure:

Males: 50 days (during 14 days pre-pairing period, and 36 days pairing period and postpairing period).
Females: Minimum 5 weeks (14 days pre-pairing period, up to 17 days pairing period, approximately 21 days of gestation period and lactation period to day 4 post partum).

Frequency of treatment:

Continuously (ad libitum)

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

2 500 ppm

Remarks:

Low Concentration

Dose / conc.:

5 000 ppm

Remarks:

Intermediate Concentration

Dose / conc.:

10 000 ppm

Remarks:

High Concentration

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were selectedbased on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study no. D80882, using concentrations in feed of 5000, 10000 and 20000 ppm and resulting in adverse reduction of food consumption, body weight loss and early termination of males and females treated with feed containing test item at a concentration of 20000 ppm.A dietary inclusion level of 10000 ppm resulted in approximately 6% (males) and 10% (females) reduction in body weight compared to the control group during 15 days of treatment.
- Rationale for animal assignment (if not random): Performed after at least three days of acclimatization using a computer-generatedrandom algorithm. Bodyweights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: Once prior to the first administration of the test item and weekly thereafter.
Females: Once prior to the first administration of the test item, weekly during the pre-pairing and pairing and on days 0, 6, 13 and 20 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Once during acclimatization, at three-day intervals during pre-pairing and postpairing periods and weekly during pairing period.
Females: Once during acclimatization, at three-day intervals during pre-pairing period, weekly during pairing period and on the following days: 0, 3, 6, 9, 12, 15, 18, 21 post coitum and 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Males: At three-day intervals during pre-pairing and post-pairing.
Females: At three-day intervals during pre-pairing and gestation, and from days 1 to day 4 post partum in all females (except nos. 50, 53, 56, 58, 62, 65, 69, 74, 77, 79, 80, 82, 85, and 89 for which no food consumption was recorded during the lactation period due to a planning error). No food consumption was recorded during the pairing period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION INTAKE: Yes
- Time schedule for examinations: Daily water intake was measured gravimetrically during pre-pairing, post-pairing, gestation and lactation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All parent animals were examined macroscopically for any structural changes at the scheduled necropsy. Special attention was directed at the organs of the reproductive system. The uteri of all dams were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites and the number of implantation sites were noted. The number of corpora luteain each ovary was recorded for all pregnant females.

HISTOPATHOLOGY: Yes The tissues indicated in Table 2 and Table 3. were prepared for microscopic examination and weighed, respectively.

Parameters examined in male parental generations: [other: spermatogenesis]

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, food and water consumption, body weights, clinical laboratory investigations, organ weights, macroscopical findings, and reproduction data:
• Means and standard deviationsof various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.
• Kruskal Wallis for the righting reflex.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ruffled fur was recorded in one female of group 4 from the last day of gestation to the end of the lactation period. This was considered to be related to the treatment with the test item. No clinical signs were observed in females of groups 2 and 3 and in males at any dose level.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males - A dose-related effect of the test item on body weight development was recorded at all dose levels. During the pre-pairing period lower body weight gain was recordedin all test item-treated groups (6.2%, 4.9%, and 0.6% in groups 2, 3, and 4 on day 7) when compared to the control group (8.6%). During the post-pairing period the body weight gainwas similar in all groups. Weights were decreased in males of group 4 fromtreatment day 4 until the end of the study but remained within 10% of concurrent control group values. No significant effects on body weights were recorded in males of groups 2 and 3.

Females - A dose-related effect of the test item on body weight development was recorded at all dose levels. During the pre-pairing period lower body weight gainwas recorded in females of groups 3 and 4 (0.2%, and -3.4% on day 7) when compared to the control group (5.1%).During the gestation period the body weight gain in females of group 3 and 4 was still lower (46.9% and 37.6% vs. 56.7% in the control group on day 21). During the lactation periodno significant differences in
body weight gain were recorded. Weights were decreased in females of groups 3 and 4 from treatment day 4 until the end of the study. Body weights in females of groups 3 and 4 were within 10% of the concurrent control value during the pre-pairing period. In group 3, body weights remained at 87-93% of control for the remainder of the study. In group 4, the mean body weight fell to about 80% of the concurrent control value at the end of the gestation period and throughout the lactation period. In group 2, slightly lower body weights wererecorded during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males - Test item-related effects on food consumption were recorded in group 3 and 4. Lower food consumption (-42%) was recorded in males of group 4 during the first treatment week when compared to the control group. From treatment week 2 until the end of the treatment period the food consumption was slightly decreased (-12% during the second week of the prepairing period and -8% during post-pairing period). Slightly lower food consumption (-15%) was recorded in males of group 3 during the first treatment week. From treatment week 2 onwards the food consumption was similar to the control group. No effects were recorded in males of group 2.

Females - Test item-related effects on food consumption were recorded in group 3 and 4. Lower food consumption (-53%) was recorded in females of group 4 during the first treatment week when compared to the control group. From treatment week 2 until the end of the treatment period the food consumption was still decreased (-20% during gestation and -45% during lactation). Lower food consumption was recorded in females of group 3 during the whole treatment period (-16% during pre-paring, -14% during gestation and -37% during lactation) when compared to the control group. No effects were recorded in females of group 2.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males - A dose-related effect of the test item on food conversion efficiency was recorded at all dose levels. Lower food conversion efficiency (-55%) was recorded in males of group 4 during the prepairing period when compared to the control group. During post paring the food conversion efficiency was similar to the controls. Lower food conversion efficiency was recorded in males of groups 2 (-25%) and 3 (-31%) during the first four days of treatment when compared to the control group. Afterwards the food conversion efficiency was similar to the controls.

Females - A dose-related effect of the test item on food conversion efficiency was recorded at all dose levels. Lower food conversion efficiency was recorded in females of groups 3 and 4 during the first four days of the pre-pairing period when compared to the controls. Afterwards the food conversion efficiency was similar to the control group. At the end of the gestation lower food conversion efficiency was recorded in all test item-treated groups. During the lactation period the feed consumption in all test item-treated groups was similar to the control group.

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

effects observed, treatment-related

Description (incidence and severity):

Males - No effects of the treatment with Gum Rosin on water consumption were recorded.

Females - During the lactation period the water consumption was decreased by 33% in females of group 4 and by 20% in females of group 3. This was considered to be a test item-related effect. No effects of the treatment on water consumption were in recorded in females during the prepairing and gestation period. No effects were recorded in group 2.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects of the treatment with Gum Rosin on hematology parameters were recorded. Changes in a few parameters achieving individual statistical significance were considered to be unrelated to treatment, because they lacked a dose-relationship and/or were clearly within the historical control range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males - The following changes were considered to be test item-related:
• The activity of alkaline phosphatase was increased in males of group 3 by 48% and by 61% in group 4.
• The total bilirubin concentration was increased in males of group 4 (8.40 µmol/L vs. 0.43 in the controls).
• The creatinine concentration was increased at all dose levels (+19%, +32%, and +47% in groups 2, 3, and 4, respectively).

Increased triglyceride concentration in group 2 reaching statistical significance was considered to be incidental, because the values were within the range of the historical controls and this change was not dose-related.

Females - Increased concentration of total bilirubin in females of group 4 (3.27 µmol/L vs. 0.00 in the controls) was considered to be treatment-related. Changes in a few other parameters reaching statistical significance were considered to be incidental, because the values were within the range of the historical controls and/or the changes were not dose-related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery - None of the parameters under investigation during the functional observational battery gave an
indication of a test item-related effect.

Grip Strength - No effects of the treatment with Gum Rosin on grip strength (fore- and hind paws) were recorded.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decreased relative and absolute thymus weights recorded in females of group 4 were considered test item-related. Decreased absolute heart, thyroid and prostate weights, increased relative (to body weight) weights of brain and liver, as well as decreased relative (to brain weight) weights of heart, thyroids, prostate and kidneys in males of group 4 were consideredto be either effects caused by the lower body weights in this group or incidental findings, because they were either not dose related and/or no correlating histopathology findings were recorded. Decreased absolute kidney, and adrenal weights offemales at all dose levels, decreased absolute ovary and heart weights, and increased relative (to body weight) brain weights of group 3 and 4 females, increased relative (to body weight) weights of kidneys in group 4 females, decreased absolute pituitary and liver weights in group 4 females, as well as decreased weights relative to the brain weight of pituitary glands, liver, and spleen in group 4 females, decreased weights relative to the brain weight of heart, kidneys, adrenals and ovaries in group 3 and 4 females, decreased absolute thymus weights in group 2 and 3 females, and decreased absolute spleen weights in group 3 and 4 females were considered to be either effects caused by the lower body weights in this group or incidental findings, because they were either not dose-related and/or no correlating histopathology findings were recorded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were recorded.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In adrenals, minimal hypertrophy/vacuolation of the zona glomerulosa was recorded in 0/12, 2/12, 5/12 and 9/12 males of groups 1, 2, 3, and 4, respectively and 0/12, 0/12, 3/12 and 5/12 females of groups 1, 2, 3, and 4, respectively. The change was characterized by diffuse hypertrophy of the zona glomerulosa associated with vacuolization of the zona glomerulosa cells. Decreased lymphocytes in the cortex of the thymus was observed in 0/12, 0/12, 0/12 and 6/12 females of groups 1, 2, 3, and 4, respectively, changes being minimal, slight, moderate and marked in 3/5, 1/5, 1/5 and 1/5 females respectively. Of note, moderate focal erosion/ulcer was observed in the non-glandular gastric mucosa of rat no. 93 (group 4).

Other effects:

no effects observed

Description (incidence and severity):

No effects of the treatment with Gum Rosin on locomotor activity were recorded.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic Toxicity

Effect level:

ca. 2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic Toxicity

Effect level:

ca. 107.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: The lowest mean achvied dose level correposnding to 2500 ppm (post-pairing period)

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

other: Body weight and body weight gain

Organ:

other: Body weight

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 4. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PRE-PAIRING PERIOD
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	325	328	330	328
	ST. Dev	10.3	7.1	9.2	12.9
	N	12	12	12	12
Day 4	Mean	344	342	341	326**
	ST. Dev	10.3	7.5	9.6	10.9
	N	12	12	12	12
Day 7	Mean	353	349	346	330**
	ST. Dev	11.7	8.7	10.3	9.1
	N	12	12	12	12
Day 10	Mean	361	354	352	332**
	ST. Dev	13.0	10.7	10.4	11.3
	N	12	12	12	12
Day 13	Mean	369	367	362	342**
	ST. Dev	12.9	11.7	11.6	13.3
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PAIRING
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 2	Mean	373	367	363	338**
	ST. Dev	15.0	12.6	11.0	16.4
	N	12	12	12	12
Day 8	Mean	387	381	378	348**
	ST. Dev	15.1	13.6	14.2	18.3
	N	12	12	12	12
Day 15	Mean	400	395	393	361**
	ST. Dev	18.9	16.7	16.3	15.6
	N	12	12	12	12

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - POST-PAIRING
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	416	404	404	375**
	ST. Dev	18.6	16.8	17.5	15.5
	N	12	12	12	12
Day 4	Mean	419	411	404	379**
	ST. Dev	18.9	18.8	18.1	17.6
	N	12	12	12	12
Day 7	Mean	423	416	412	378**
	ST. Dev	18.0	19.2	16.5	17.8
	N	12	12	12	12
Day 10	Mean	425	421	415	382**
	ST. Dev	19.7	21.2	18.0	16.3
	N	12	12	12	12
Day 13	Mean	425	423	418	385**
	ST. Dev	19.2	22.9	18.1	16.0
	N	12	12	12	12
Day 16	Mean	428	425	419	383**
	ST. Dev	18.9	23.9	18.3	17.7
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - PRE-PAIRING PERIOD
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	219	220	217	216
	ST. Dev	9.3	8.6	8.4	8.0
	N	12	12	12	12
Day 4	Mean	227	227	215**	206**
	ST. Dev	7.3	13.2	8.1	9.2
	N	12	12	12	12
Day 7	Mean	230	231	217*	209**
	ST. Dev	8.4	13.9	9.1	10.9
	N	12	12	12	12
Day 10	Mean	235	232	222*	211**
	ST. Dev	10.0	13.8	9.1	10.3
	N	12	12	12	12
Day 13	Mean	237	236	225*	213**
	ST. Dev	11.7	15.2	7.3	12.1
	N	12	12	12	12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 8. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - GESTATION
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 0	Mean	245	242	229**	220**
	ST. Dev	11.0	12.7	9.6	9.8
	N	12	10	11	11
Day 3	Mean	262	257	243**	230**
	ST. Dev	10.6	13.7	11.7	11.0
	N	12	10	11	11
Day 6	Mean	274	267	252**	239**
	ST. Dev	11.2	14.0	13.6	9.9
	N	12	10	11	11
Day 9	Mean	283	277	260**	247**
	ST. Dev	10.9	13.2	13.9	11.4
	N	12	10	11	11
Day 12	Mean	297	292	272**	256**
	ST. Dev	14.1	13.0	17.9	10.3
	N	12	10	11	11
Day 15	Mean	314	306	287**	270**
	ST. Dev	14.9	15.3	17.0	13.7
	N	12	10	11	11
Day 18	Mean	347	343	315**	291**
	ST. Dev	21.7	15.0	19.7	13.7
	N	12	10	11	11
Day 21	Mean	385	368	336**	302**
	ST. Dev	28.6	20.5	23.4	15.8
	N	12	10	11	11

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 9. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - LACTATION
		Group 1 0 ppm	Group 2 2500 ppm	Group 3 5000 ppm	Group 4 10000 ppm
Day 1	Mean	282	263*	250**	232**
	ST. Dev	20.5	15.5	20.1	12.3
	N	11	11	11	11
Day 4	Mean	296	280*	257**	237**
	ST. Dev	18.0	18.3	15.1	11.8
	N	12	11	11	11

* / **: T-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. Test item intake of Males during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	151.5
P	5000	285.2
P	10000	472.9

 

Table 11. Test item intake of Males during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0
P	2500	107.7
P	5000	209.9
P	10000	447.0

 

Table 12. Test item intake of Males during post-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	107.7
P	5000	209.9
P	10000	447.0

 

Table 13. Test item intake of Females during pre-pairing period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	174.9
P	5000	313.7
P	10000	522.6

 

 Table 14. Test item intake of Females during Gestation Period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	183.4
P	5000	349.2
P	10000	693.2

 

 Table 15. Test item intake of Females during lactation period:

Generation	Concertation (ppm)	Mean Achieved Dose Level (mg/kg bw/day)
P	0	0
P	2500	231.0
P	5000	377.6
P	10000	691.9

 

Conclusions:

Based on the results of this study, a NOEL (No Observed Effect Level) for general toxicity could not be established due to effects on body weights at all dose levels in females during the lactation period. The NOAEL (No Observed Adverse Effect Level) for general toxicity was established at the dose level of 2500 ppm. For males, the lowest mean achived dose level of 107.7 mg/kg/bw was derived at concetration 2500 ppm.

Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity study, Gum Rosin was administered to rats (12/sex/dose) in dietary mixtures at concentrations of 0, 2500, 5000, and 10000 ppm for a period of 50 days for male rats and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (at least 36 days, up to 53 days).

The treatment with the test item did not lead to any premature mortalities and had no effects on behaviour, reflexes, grip strength, locomotor activity, and haematology parameters. 

In the high dose group (10000 ppm), in both sexes, significantly lower food consumption was recorded during the first treatment week. From treatment week 2 until the end of the treatment period the food consumption was slightly decreased in males, but still significantly decreased in females when compared to the control group. The lower food consumption led to a transient lower body weight gain and lower body weights until the end of the study in both sexes. Except for ruffled fur in one female from the last day of gestation to the end of the lactation period, no clinical signs were observed. The water consumption was significantly reduced during the lactation period which was possibly due to the lower number of pups and therefore lower milk production in this group.

Increased activity of alkaline phosphatase in males and increased bilirubin concentration in both sexes did not correlate with other findings and were therefore not considered adverse. Decreased absolute and relative thymus weights in females recorded at necropsy correlated with decreased lymphocytes in the cortex of the thymus. This finding was considered to be stress-related and likely not a direct effect of the test item.

In the intermediate dose group (5000 ppm), lower food consumption was recorded in males and females during the first treatment week. From treatment week 2 onwards the food consumption was similar to the control group in males, but still decreased in females. The lower food consumption led to a transient lower body weight gain in males during the pre-paring period. However, decreased body weights compared to the concurrent controls were recorded during the entire treatment period in females. No clinical signs were observed at this dose level. The water consumption of females was slightly reduced during the lactation period.

In the low dose group (2500 ppm), a transiently slightly lower body weight gain was recorded during the pre-pairing period in males. Afterwards the body weight development at this dose level was similar to the controls.

The creatinine concentration was increased at all dose levels in males. An increase of creatinine is usually a sign for kidney damage. However, no histopathological findings were recorded in the kidneys. Therefore, the increase of the creatinine was not considered to be an adverse effect. Minimal hypertrophy/vacuolation of the zona glomerulosa was observed in the adrenal glands of males from the 2500 ppm group and in females from the 5000 ppm group with dose related-incidence. The pathogenesis of this change is uncertain. The zona glomerulosa is the site of synthesis of aldosterone which is mainly involved in the control of salt and water balance in the body. Secretion of aldosterone is controlled through the renin-angiotensin system and hypertrophy of the zona glomerulosa is generally considered to be an adaptative process following stimulation of this system. Therefore, this change was not considered to be an adverse effect.

Based on the results of this study, a NOEL (No Observed Effect Level) for general toxicity could not be established due to effects on body weights at all dose levels in females during the lactation period. The NOAEL (No Observed Adverse Effect Level) for general toxicity was established at the dose level of 2500 ppm. For males, the lowest mean achieved dose level of 107.7 mg/kg/bw was derived at concertation 2500 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

107.7 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Justification for classification or non-classification

Not classified for specific target organ toxicity – repeated exposure according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 orUN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).