Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Endpoint summary

Description of key information

The NOAEL for systemic toxicity in a dietary OECD TG 422 is > 717 and > 1175 mg/kg bw for males and females, respectively (equivalent to 7500 ppm nominal) and therefore it can be concluded that no adverse effects are seen.

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:

experimental study

Adequacy of study:

key study

Study period:

March-July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 10 weeks, females 13 weeks
- Weight at study initiation: males 235 to 307 g; females 199 to 246 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA) without cage enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: males 8 days, females 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-21.6
- Humidity (%): 45.4-70.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 March 2017 To: 01 June 2017

Route of administration:

oral: feed

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) for a maximum of 8 days until use, if not used on the day of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.
-concentration analysis
Duplicate samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
-homogeneity analysis
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. After acceptance of the analytical results, backup samples were discarded.
-stability analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the experimental conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated for 28 days, up to and including the day before scheduled necropsy
Females that delivered offspring were treated for 50 - 55 days; Females that failed to deliver healthy offspring were treated for 41 days.

Frequency of treatment:

continuous (by feed)

Dose / conc.:

500 other: ppm (equivalent to 30 mg/kg bw nominal)

Dose / conc.:

2 000 other: ppm (equivalent to 130 mg/kg bw nominal)

Dose / conc.:

7 500 other: ppm (equivalent to 500 mg/kg bw, nominal)

No. of animals per sex per dose:

10 (and 3 additional high dose males to determine sperm parameters after 4 days of administration)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the chemical properties of the test item which becomes an acid upon administration because the ester is cleaved with the potential for acidification of the blood, on the results of a 14-day oral range finding study with dietary administration of Veramoss in the rat, and in an attempt to produce graded responses to the test item.
In the 14 day study the NOAEL was 7500 ppm corresponding to 758 mg/kg bw/day for males and 735 mg/kg bw/day for females which was the highest dose level tested (in consultation with the Sponsor). No toxicologically significant changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, organ weights and microscopic examination.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters according to Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters according to guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: week 4 of treatment (males) or last week of lactation (females)
- How many animals: 5 per group
- Dose groups that were examined: all
- Parameters checked: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity.

OTHER: sperm analysis from group 4 additional males after 4 days of dietary administration; plasma levels of T4 in F0 males and PND 14-15 pups, estrous cycle determination F0 females.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to Guideline

HISTOPATHOLOGY: Yes, according to Guideline

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

Two females were sacrificed unscheduled on PND 1 (one Group 1 and one Group 4 female) due to aggressive behavior and cannibalism of their healthy pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Females at 500 ppm and 7500 ppm showed somewhat lower body weight gain during the post-coitum period (statistically significant on Day 14 and Day 17 for 500 ppm females and Day 14 for 7500 ppm females), resulting in 5% and 7% lower mean body weights on Day 20 post-coitum (not statistically significant) respectively. Thereafter, during lactation, mean body weights of 7500 ppm females remained lower compared to controls (up to 10%), reaching statistical significance at lactation Days 4 and 7 (the difference on lactation Day 13 was smaller and not statistically significant due to the poor weight gain of control females between Days 7-13). Body weight gain of 7500 ppm females during lactation was normal. Body weight and body weight gain of females treated up to 2000 ppm and males treated up to and including 7500 ppm were unaffected by treatment.
The statistically significant increase in body weight gain noted in females at 2000 ppm during lactation (Day 13) were considered unrelated to treatment due to the lack of a dose-related response and/or relatively low control value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
At 7500 ppm, food consumption of males was higher compared to controls on several days during the treatment period, resulting in a higher overall mean food consumption for the premating phase (relative difference from controls: 15%). Females at 7500 ppm consumed less food than controls on the first treatment day. Thereafter, their food consumption generally remained close to control values (for one cage of the 7500 ppm females (cage no. 18) food consumption values were higher compared to controls on a few days during the pre-mating period). Overall, food consumption was considered not to be affected by treatment since no consistent differences from controls were noted. The statistically significant differences in females at 2000 and 7500 ppm noted on a single occasion during the post-coitum period (Days 22-23 when group means were based on only a few females) and the lactation period (Days 9-10) and for 7500 ppm the food consumption corrected for body weight in the lactation period (Days 12-13) were considered to be chance findings.
The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table in "any other information"

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters (red and white blood cells) were considered not to have been affected by treatment.
Isolated statistically significant variations noted in red blood cell parameters (slightly lower hemoglobin and hematocrit in females at 500 and 7500 ppm) were considered unrelated to treatment due to the absence of a dose-related trend.
Coagulation parameters were considered not to have been affected by treatment.
An isolated statistically significant variation noted in prothrombin time (lower in males at 2000 ppm) was considered unrelated to treatment due to the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant differences were noted between the 7500 ppm group and the control group:
• Higher potassium in males (1.09 x of control). Values in 7500 ppm males and in one control male were at or slightly above the upper limit of the historical control range. (Historical control data period 2015-June 2017; potassium in male rats (mmol/L) mean = 3.94, P5-P95 = 3.54-4.37 (n=219))
• Lower total protein in females (0.92 x of control). Values in 7500 ppm females remained within the historical control range. (Historical control data period 2015-June 2017; total protein in female rats (g/L) mean = 61.8, P5-P95 = 54.7-68.1 (n=186))
In females a large variety in bile acids were noted, both in the control group and 7500 ppm females this is caused by an individual animal with a markedly higher value compared to the remainder of the group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. In the absence of a dose-related response, the statistically significantly lower fore limb grip strength in females at 500 ppm was considered unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Thyroid weight for 2000 ppm females was statistically significant decreased compared to controls; this was considered unrelated to treatment due to the absence of a dose-related trend.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 717 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

Key result

Critical effects observed:

no

Diet analyses

Accuracy: No test item was detected in the control diet.

The concentrations analyzed in the 500, 2000 and 7500 ppm diets were in agreement with target concentrations, with mean accuracies between 97% and 100%).

Homogeneity: The 500 ppm and 7500 ppm diets were homogeneous (coefficient of variation 2.9 and 5.0%, respectively).

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the text table below.

		Mean over means intake[mg test item/kg body weight/day] (mean range indicated between brackets)
sex	Study period	500 ppm	2000 ppm	7500 ppm
Males	Pre-mating	45 (41-51)	197 (168-238)	790 (623-1062)
	Post-mating	35 (31-47)	150 (134-238)	590 (500-819)
	Mean of means^a	41	181	717
Females	Pre-mating	42 (36-59)	177 (145-238)	737 (471-1035)
	Post-coitum	64 (44-76)	235 (116-296)	995 (648-1217)
	Lactation	113 (73-144)	472 (305-593)	1907 (1116-2514)
	Mean of means^a	71	281	1175

aMean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14x mean pre-mating) + (13x mean post-mating)) / 27

Females (Group 2 and 3): ((14 x mean pre-mating) + (26 x mean post-coitum) + (14 x mean lactation)) / 54

Females (Group 4): ((14 x mean pre-mating) + (23 x mean post-coitum) + (14 x mean lactation)) / 51

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 7500 ppm was established, which corresponds to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively. These values are very close to the limit dose of 1000 mg/kg bw and therefore the final conclusion is that 'no adverse effects are observed'.

Executive summary:

Introduction: The objectives of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) were to determine the potential toxic effects of the substance when given via diet for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) no-observed-adverse-effect levels (NOAELs) were evaluated. In this section only the repeated dose parameters are presented. The fertility and developmental effects are presented in the reproductive toxicity sections.

Method: The dose levels in this study were based on the results of a dose range finder. Rats (10/sex) received food with vehicle or test substance at levels of 500, 2000 or 7500 ppm (nominal 30, 130 and 500 mg/kg bw/day, respectively).

Results: Accuracy and homogeneity of diet preparations were demonstrated by analyses. The intake of the substance was higher than expected and resulted in an overall average for the high dose of 717 mg/kg bw for males and 1175 mg/kg bw for females.

Clinical signs: Exposure to the test item up to 7500 ppm was well tolerated as indicated by the absence of adverse changes in the parental parameters examined in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations).

Body weight gainof females at 7500 ppm was somewhat reduced during lactation. As the associated decreases in mean body weight did not exceed 10% this finding was considered not to be adverse.

Heamatology: No adverse effect seen.

Clinical biochemistry: No adverse effects seen. A higher mean plasma level of potassium was noted in males treated at 7500 ppm. This change was regarded as non-adverse based on its modest magnitude (9% difference from controls) and the absence of changes indicative of target organ toxicity or alterations in the general function of the animals. A lower mean plasma level of total protein was observed in females treated at 7500 ppm. This change was regarded as non-adverse due to its slight magnitude (8%, within the historical control range, see results section) and the absence of any evidence of conditions known to be associated with decreases in total protein.

Organ effects: Absolute and relative weights, macroscopically and microscopically there were no adverse effect seen.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental no-observed-adverse-effect level

(NOAEL) of at least 7500 ppm was established (corresponding to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Mode of Action Analysis / Human Relevance Framework

Veramoss is a low molecular weight ester which will hydrolyse and metabolise into its acid in the gut, resulting in a aromatic acid with two additional hydroxyl groups. This relatively strong acid likely will be distributed as such to the kidneys and limited liver metabolisation is expected, which is supported with absence of liver weight increase usually occurring upon addition of a xenobiotic and its metabolic demand.

Additional information

Introduction: The objectives of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) were to determine the potential toxic effects of the substance when given via diet for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) no-observed-adverse-effect levels (NOAELs) were evaluated. In this section only the repeated dose parameters are presented. The fertility and developmental effects are presented in the reproductive toxicity sections.

Method: The dose levels in this study were based on the results of a dose range finder. Rats (10/sex) received food with vehicle or test substance at levels of 500, 2000 or 7500 ppm (nominal 30, 130 and 500 mg/kg bw/day, respectively).

Results: Accuracy and homogeneity of diet preparations were demonstrated by analyses. The intake of the substance was higher than expected and resulted in an overall average for the high dose of 717 mg/kg bw for males and 1175 mg/kg bw for females.

Clinical signs: Exposure to the test item up to 7500 ppm was well tolerated as indicated by the absence of adverse changes in the parental parameters examined in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations).

Body weight gainof females at 7500 ppm was somewhat reduced during lactation. As the associated decreases in mean body weight did not exceed 10% this finding was considered not to be adverse.

Heamatology: No adverse effect seen.

Clinical biochemistry: No adverse effects seen. A higher mean plasma level of potassium was noted in males treated at 7500 ppm. This change was regarded as non-adverse based on its modest magnitude (9% difference from controls) and the absence of changes indicative of target organ toxicity or alterations in the general function of the animals. A lower mean plasma level of total protein was observed in females treated at 7500 ppm. This change was regarded as non-adverse due to its slight magnitude (8%, within the historical control range, see results section) and the absence of any evidence of conditions known to be associated with decreases in total protein.

Organ effects: Absolute and relative weights, macroscopically and microscopically there were no adverse effect seen.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental no-observed-adverse-effect level

(NOAEL) of at least 7500 ppm was established (corresponding to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively).

Justification for classification or non-classification

Based on the absence of adverse effects in a reliable repeated dose toxicity study with the reproduction/developmental screening test the substance does not have to be classified for repeated dose toxicity by the oral route according to EU CLP (EC No. 1272/2008 and its amendments).