Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with international guidelines in a GLP laboratory. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-1,3,5-trimethyl-1,3,5-triazine

EC Number:

203-612-8

EC Name:

Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Cas Number:

108-74-7

Molecular formula:

C6H15N3

IUPAC Name:

1,3,5-trimethyl-1,3,5-triazinane

Test material form:

other: solution

Details on test material:

Identification: Hexahdyro-1,3,5-trimethyl-1,3,5-triazineAppearnace: Clear, colourless liquidTest item Storage: At room temperatureCAS Number: 108-74-7Molecular Formula: C6H15N3

Test animals

Species:

rat

Strain:

other: Rat: Crl: WI (HAN) (outbred, SPF-Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Age at study initiation: Approximately 10-14 weeks- Fasting period before study: no- Housing: Females were individually housed in Macrolon cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage enrichment were supplied.General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. - Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). - Water (e.g. ad libitum): Free access to tap-water. - Acclimation period: At least 5 days prior to start of treatment.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 ± 3°C (actual range: - Humidity (%): 40 - 70% (actual range: - Air changes (per hr): 10- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 10 January 2016 (start treatment) To: 28 January 2016 (end of in-life phase)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for the density of the test substance (1.0236), the specific gravity of the vehicle (1.036) and the water content (58.4249%).VEHICLE- Based on trial formulations performed at WIL Research Europe B.V.- Dose volume: 5 mL/kg bodyweight, experimental dose levels of neat Hexahydro-1,3,5-Trimethyl-1,3,5-Triazine was 0, 10, 40 and 120 mg hexahydro-1,3,5-trimethyl-1,3,5-triazine/kg b.w.- Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on two occasions during the treatment phase (11 January (analysed the same day) and 26 January (refrigerated and analysed the day after) 2016), according to a validated method (ABL study no. 15342; WIL project no. 511297). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature was also determined (highest and lowest concentration) on one occasion.The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

Untreated females were mated at the supplier and were at Day 0 or 1 post coitum on arrival at the test facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).

Duration of treatment / exposure:

Rats were treated for 14 days between days 6 to 20 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose over the complete treatment period

Duration of test:

23 days including mating of females at supplier and necrospy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection was based on a 28-day repeated dose toxicity study, (Wil project 491807), a dose range finding study (WIL project 491831) and an acute oral toxicity study (NOTOX project 491805).

Examinations

Maternal examinations:

Parental animals: Observations and examinationsMORTALITY/VIABILITY OBSERVATIONS: Yes - Time schedule: At least twice daily (early morning/late afternoon)DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: At least once daily 1 hour after dosing (+/- 30 minutes) from Day 2 post-coitum onwards up to the day prior to necropsy. At least once daily 1 hour after dosing (± 30 minutes) from Day 2 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. BODY WEIGHT: Yes- Time schedule for examinations: Females were weighed on Days 2, 6, 9, 12, 15, 18 and 21 post-coitumFOOD CONSUMPTION: YesFood consumption was measured on Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitumWATER CONSUMPTION: No, subjective appraisal will be maintained during the study, but no quantitative assessment was introduced as no treatment related effect was suspected.NECROPSY EXAMINATIONS: All animals were sacrificed on Day 21 post-coitum using an oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).The stomach and the eyes of all animals were dissected and examined. The stomach was fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). The eyes were fixed in modified Davidson’s solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial; all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:-The number of corpora lutea.-The weight of the (gravid) uterus.-The number and distribution of live and dead fetuses.-The number and distribution of embryo-fetal deaths-The weight of each fetus.-The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).-Externally visible macroscopic fetal abnormalities. In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).EXTERNAL: All viable fetuses were examined, weighed and sexed. All live fetuses were euthanized by administration of 0.5 mL of sodium pentobarbital (Euthasol(R) 20 %, AST Farma B.V., Oudewater) into the oral cavity using a small metal or plastic feeding tube. For late resoprtions a gross external examination was performed. Late resorptions were discarded. VISCERAL (INTERNAL): Approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was confirmed by internal examination.The heads were removed from these fetuses and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination. After examination, the tissues without variations or malformations were discarded. Tissues with variations or malformations were stored in 10% buffered formalin.Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded. The carcasses were processed and stained with Alizarin Red S (as described below (SKELETAL)), but not examined in first instance.SKELETAL:From the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96 % aqueous ethanol, macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands). Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

The following statistical methods were used to analyze the data:-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.-The Fisher Exact-test was applied to frequency data.-The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.-Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. 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 might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Piloerection was noted during treatment for eight females at 40 mg/kg and six females at 120 mg/kg. As it was seen for 1-2 days only and also occurred during pretest for some animals, it was not considered toxicologically relevant. Other signs of toxicity seen in the treatment groups (i.e. uncoordinated movements, hunched posture and rales) were observed for single animals for up to 3 days only, and therefore not considered to be related to treatment.Incidental findings that were noted included alopecia, scabs, scales, erythema and salivation. As these findings occurred within the range of background findings observed in rats of this age and strain under the conditions in this study and were not consistent over time, they were considered not to be toxicologically significant.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related microscopic findings:Stomach (Glandular):- Secretory depletion was present in 6/10 females treated at 120 mg/kg/day up to slight degree.- Inflammatory cell infiltrate, granulocytic was present at increased incidence and severity in 7/10 females treated at 120 mg/kg/day up to slight degree, compared to a single female at a minimal or slight degree in the remaining dose groups.The remainder of the recorded microscopic findings, including those in the eyes, 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

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Relatively high mean value for post-implantation loss (7.4% per litter) was noted at 10 mg/kg, compared to the control group and the other treatment groups. This was primarily the result of the high values in two individual females. The post-implantation values for the other females in the 10 mg/kg group were in the same range at noted for the other groups. As no dose-dependent relationship was observed, this was considered as incidental and not treatment-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One female in the 40 mg/kg group was non-pregnant. All pregnant females had litters with viable fetuses.

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects: yes. Remark: some microscopic, none toxicologically relevant effects were observed. Details on maternal toxic effects: One female was non-pregnant (at 40 mg/kg bw) All females had litters with viable fetuses. In comparison to the control group the 10 mg/kg bw group had a relatively high mean value for post-implantation loss (7.4 % per litter). This was due to high values in two individual females. No dose-dependent relationship was observed, this was considered as incidental. MACRO- AND MICRO-SCOPIC EXAMINATION: Macroscopic examination at necropsy revealed no treatment-related findings. One female at 120 mg/kg showed an interrupted left horn of the uterus. This was considered as incidental and not toxicologically significant. One control female showed alopecia. There were test item-related microscopic findings: Stomach (Glandular):-Secretory depletion was present in 6/10 females treated at 120 mg/kg/day up to slight degree.-Inflammatory cell infiltrate, granulocytic was present at increased incidence and severity in 7/10 females treated at 120 mg/kg/day up to slight degree, compared to a single female at a minimal or slight degree in the remaining dose groups. The remainder of the recorded microscopic findings, including those in the eyes, 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

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to 120 mg/kg.Mean combined (male and female) fetal body weights were 5.0, 5.1, 5.2 and 5.2 for the control, 10, 40 and 120 mg/kg groups, respectively.The statistically significant changes noted for female fetal body weights at 40 and 120 mg/kg were not considered toxicologically relevant as the increase was very slight, did not show a dose response relationship, and all values were within normal limits.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 120 mg/kg.Skeletal malformations noted in this study were bent limb bones (Group 2 fetus A035-01 and control fetuses A013-01 and -03 of the same litter) and a rib anomaly (Group 2 fetus A043-01). Due to the single occurrence at the low dose level and/or occurrence in control fetuses, these malformations were not considered to be treatment related.The variation of 14th rudimentary ribs was noted at an incidence of 71.8%, 54.1%, 50.4% and 48.0% per litter in Groups 1, 2, 3 and 4, respectively. Compared with the current control incidence, the incidences in Groups 3 and 4 were statistically significantly reduced. From historical control data it appears that the incidence of 14th rudimentary ribs ranged from 19.0% to 72.0% per litter. In comparison with this, the current control value lay at the maximum historical control value, while thevalues of all treated groups lay closer the historical mean value of 44.1% per litter. Taken this into account, it was considered that the striking group distribution of 14th rudimentary ribs in this study occurred by chance and was not related to treatment.Other skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend and/or occurred infrequently.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Apart from a fetus at 40 mg/kg (A061-02), which had situs inversus whereby all organs were laterally transposed, there were no other viscerally malformed fetuses observed.The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, discolored adrenal and liver, partially undescended thymus horn(s), convoluted ureter and retroesophageal right subclavian artery. These variations occurred at low incidences and/or in the absence of a dose-related incidence trend and therefore were not considered to be treatment related.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no treatment related effects on any litter size for any test group as compared to the control. All litter sizes were 11 +/- 0.5. Sex ratio was unaffected by treatment and were ~50:50 for all treatment and control groups. There were no effects on foetal bodyweight and these were 5.0 +/- 0.2 g. MORPHOLOGICAL EXAMINATIONS: EXTERNAL: There were no treatment related effects on external morphology following treatment up to 120 mg/kg. The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group. There were no treatment related effects on external morphology following treatment up to 120 mg/kg. The only malformation in this study was noted in a fetus at 40 mg/kg (A046-02 had an omphalocele) and external variations were not seen in any group. No dose-dependent relationship was observed, this was considered as incidental. VISCERAL: There were no treatment related effects on visceral morphology following treatment up to 120 mg/kg. Apart from a fetus at 40 mg/kg (A061-02), which had situs inversus whereby all organs were laterally transposed, there were no other viscerally malformed fetuses observed. The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, discolored adrenal and liver, partially undescended thymus horn(s), convoluted ureter and retroesophageal right subclavian artery. These variations occurred at low incidences and/or in the absence of a dose-related incidence trend and therefore were not considered to be treatment related. SKELETAL: There were no treatment related effects on skeletal morphology following treatment up to 120 mg/kg. Skeletal malformations noted in this study were bent limb bones (Group 2 fetus A035-01 and control fetuses A013-01 and -03 of the same litter) and a rib anomaly (Group 2 fetus A043-01). Due to the single occurrence at the low dose level and/or occurrence in control fetuses, these malformations were not considered to be treatment related. The variation of 14th rudimentary ribs was noted at an incidence of 71.8%, 54.1%, 50.4% and 48.0% per litter in Groups 1, 2, 3 and 4, respectively. Compared with the current control incidence, the incidences in Groups 3 and 4 were statistically significantly reduced. From historical control data it appears that the incidence of 14th rudimentary ribs ranged from 19.0% to 72.0% per litter. In comparison with this, the current control value lay at the maximum historical control value, while the values of all treated groups lay closer the historical mean value of 44.1% per litter. Taken this into account, it was considered that the striking group distribution of 14th rudimentary ribs in this study occurred by chance and was not related to treatment. Other skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend and/or occurred infrequently. CONCLUSIVE REMARK No toxicologicallly relevant effects were observe.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No developmental toxicity observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for hexahydro-1,3,5-trimethyl-1,3,5-triazine was established as being 40 mg/kg for local toxicity based on microscopic findings in the stomach. The maternal NOAEL for systemic toxicity was at least 120 mg/kg.The developmental NOAEL was established as being at least 120 mg/kg.

Executive summary:

Study outline

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 10, 40 and 120 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on two days during treatment were analyzed for accuracy, homogeneity and/or stability.

 

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortemand external, thoracic and abdominal macroscopic findings were recorded. Stomach and eyes were collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach and eyes from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

 

RESULTS

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

 

Maternal findings

No maternal toxicity was observedin the 10, 40 and 120 mg/kg groups.

 

There were test item-related microscopic findings in the stomach (glandular) at 120 mg/kg. Findings consisted of secretory depletion and inflammatory cell infiltrate, both up to slight degree.

Developmental findings

No developmental toxicity was observed in the 10, 40 and 120 mg/kg groups.

 

CONCLUSION

 

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Effect Level (NOEL) for Hexahydro-1,3,5-trimethyl-1,3,5-triazine was establishedas being 40 mg/kg for local toxicity based on microscopic findings in the stomach. The maternal NOAEL for systemic toxicity was at least 120 mg/kg.

 

The developmental toxicity was established as being over or equal to 120 mg/kg (the highest dose tested).